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Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

In systemic mastocytosis (SM), qualitative and serial quantitative assessment of the KIT D816V mutation is of diagnostic and prognostic relevance. We investigated peripheral blood and bone marrow samples of 161 patients (indolent SM (ISM), n = 40, advanced SM, AdvSM, n = 121) at referral and during follow-up for the KIT D816V variant allele frequency (VAF) at the DNA-level and the KIT D816V expressed allele burden (EAB) at the RNA-level. A round robin test with four participating laboratories revealed an excellent correlation (r &gt, 0.99, R2 &gt, 0.98) between three different DNA-assays. VAF and EAB strongly correlated in ISM (r = 0.91, coefficient of determination, R2 = 0.84) but only to a lesser extent in AdvSM (r = 0.71, R2 = 0.5). However, as compared to an EAB/VAF ratio ≤2 (cohort A, 77/121 patients, 64%) receiver operating characteristic (ROC) analysis identified an EAB/VAF ratio of &gt, 2 (cohort B, 44/121 patients, 36%) as predictive for an advanced phenotype and a significantly inferior median survival (3.3 vs. 11.7 years, p = 0.005). In terms of overall survival, Cox-regression analysis was only significant for the EAB/VAF ratio &gt, 2 (p = 0.006) but not for VAF or EAB individually. This study demonstrates for the first time that the transcriptional activity of KIT D816V may play an important role in the pathophysiology of SM.

Radiation-induced bystander effects (RIBE) in human hematopoietic stem and progenitor cells may initiate myeloid neoplasms (MN). Here, the occurrence of RIBE caused by genotoxic signaling from irradiated human mesenchymal stromal cells (MSC) on human bone marrow CD34+ cells was investigated. For this purpose, healthy MSC were irradiated in order to generate conditioned medium containing potential genotoxic signaling factors. Afterwards, healthy CD34+ cells from the same donors were grown in conditioned medium and RIBE were analyzed. Increased DNA damage and chromosomal instability were detected in CD34+ cells grown in MSC conditioned medium when compared to CD34+ cells grown in control medium. Furthermore, reactive oxygen species and distinct proteome alterations, e.g., heat-shock protein GRP78, that might be secreted into the extracellular medium, were identified as potential RIBE mediators. In summary, our data provide evidence that irradiated MSC induce genetic instability in human CD34+ cells potentially resulting in the initiation of MN. Furthermore, the identification of key bystander signals, such as GRP78, may lay the framework for the development of next-generation anti-leukemic drugs.

Background: In general, patients with hematological diseases are predisposed to develop infections. Severe COVID-19 infection associated with high mortality is more likely in these patient cohorts compared to the general population. Due to immune defects related to the primary disease and/or to immunosuppressive treatment regimes, vaccination efficacy may be reduced in patients with hematological diseases. So far, data on this area are limited. Aim: To evaluate vaccination-related antibody response to BNT162b2, mRNA-1273, and ChADOx1 in patients with hematological disorders. Patients and methods: In this interim analysis of a prospective, observational single-center study, we report antibody levels at least 2 weeks after COVID-19 vaccination. A FDA/CE approved electrochemiluminescent assay (ECLIA) (Elecsys®, Roche, Mannheim, Germany) was used to quantify antibodies, pan Ig (including IgG) against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The assay has a measurement range of 0.4 to 250 U/mL, with a concentration ≥0.8 U/ml considered as positive. Data were analyzed for patients without detection of anti-N (nucleocapsid) SARS-CoV-2 antibody (i.e., without having passed SARS-CoV-2 infection). All tests were performed according to the manufacturer's instructions in an accredited laboratory at the University Hospital Mannheim. Results: Between February 2021 and July 2021, a total of 175 patients with hematological diseases were included in this study. The median age was 66 years (range 21-90 years), and 81 (46.3%) were female. The antibody levels were measured at least 14 days (median, 58 days) after the 2 nd vaccination. The patients were vaccinated with BNT162b2 (BioNTech, n=134), mRNA-1273 (Moderna, n=19), ChADOx1 (AstraZeneca, n=12), or got the first vaccination with BNT162b2 and the second with ChADOx1 (n=10). Overall, 145/175 (82.9%) were diagnosed with a malignant hematological disease (myeloid neoplasms, n=108; lymphoid neoplasms, n=37) and 30/175 with a non-malignant hematological disease (autoimmune disease, n=24; benign, n=6). 124 patients (70.1%) were on active therapy, and 51 patients (29.1%) were previously treated or treatment naïve. Correlation to specific therapies is ongoing and will be presented. In general, vaccination-related antibody response was positive (≥0.8 U/mL) in 148/175 (84.6%) patients with a median level of 208.6 U/mL (range 0.8-250.00) and negative ( In myeloid neoplasms, patients with classical myeloproliferative neoplasm (MPN) had the highest negative result for antibodies with 4/7 (57.1%) followed by myelodysplastic syndrome (MDS) 2/7 (28.6%). Interestingly, all patients with chronic myeloid leukemia (CML) had a measurable immune response. In lymphoid neoplasms, patients with low-grade non-hodgkin lymphoma (NHL) (predominately chronic lymphocytic leukemia, CLL) had the highest negative antibody result 13/16 (81.3%) followed by high-grade NHL 4/8 (50%; predominately diffuse large b-cell lymphoma, DLBCL). In non-malignant hematological diseases, only patients with autoimmune diseases had a negative result. Conclusion: A remarkable group of patients with hematological disease were measured with no or low immune response after 2 nd COVID-vaccination, especially those with low-grade NHL, MDS and autoimmune disease. It seems that the percentage of patients with MPN and low response is less critical. No problems appeared in CML patients. Further explorations are needed with focus on potential risk of COVID infections despite full vaccination: The potential of 3 rd booster vaccination should be explored within clinical trials. Disclosures Reiter: AOP Orphan Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Abbvie: Membership on an entity's Board of Directors or advisory committees; Deciphera: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses. Kreil: Novartis: Research Funding. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Jawhar: Takeda: Honoraria, Other: Travel support; Blueprint Medicines: Honoraria; Stemline: Consultancy, Honoraria; Celgene: Other: Travel support; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Saussele: Roche: Honoraria; Pfizer: Honoraria; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Systemic mastocytosis (SM) is characterized by activating mutations in KIT, usually KIT D816V in >90% of patients. According to the WHO classification, detection of KIT D816V is a minor diagnostic criterion. Moreover, a reduction of the RNA-based KIT D816V expressed allele burden (EAB) of ≥25% at month 6 is a favorable marker for improved survival in midostaurin-treated advanced SM (AdvSM) patients. However, only limited data exist upon the comparison between RNA-based and DNA-based quantitative KIT D816V mutation analyses. We therefore collected peripheral blood samples from 161 SM patients (indolent SM [ISM], n=40; AdvSM, n=121) at time of referral. We applied a real time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay for assessment of the KIT D816V EAB and a chip-based digital PCR (dPCR) for the quantification of the DNA-based KIT D816V variant allele frequency (VAF, QuantStudio 3D dPCR System, ThermoFisher Scientific, Massachusetts, USA). The limit of detection (LOD) was assessed through serial dilution experiments with DNA from healthy individuals and DNA from a SM patient with a KIT D816V VAF of approximately 50%. All samples were analyzed in two independent dPCR runs. In an inital round-robin test for an inter-laboratory (n=4) comparison on 30 DNA samples (ISM, n=7; AdvSM, n=19), we found a very good correlation between 3 different chip-based digital PCR systems (dPCR; droplet-based dPCR, ddPCR, BioRad Laboratories, California, USA; quantitative real-time PCR, qPCR, California, USA; dPCR vs. ddPCR r=0.99, R2=0.99; dPCR vs. qPCR r=0.99, R2=0.98). In ISM patients, the comparison between EAB and VAF revealed a strong linear relationship with a correlation (r) of 0.91 and a coefficient of determination (R2) of 0.84, which was significantly inferior (r=0.71; R2=0.5) in AdvSM patients. In 45/121 (37%) and 76/121 (63%) of AdvSM patients, the EAB/VAF ratio was ≤2 (cohort A) or >2 (cohort B). To confirm the significant disparity between EAB and VAF in individual patients, serial analyses of at least 3 samples in 12 patients revealed a stable EAB/VAF ratio during follow-up. The comparison between cohort A and cohort B revealed significant differences in terms of a higher median hemoglobin level (p=0.006), a lower percentage of patients with hemoglobin 2 present with an aggressive phenotype and adverse prognosis as compared to patients with EAB/VAF ratio ≤2. We therefore recommend to routinely determine KIT D816V EAB and VAF in AdvSM patients. Disclosures Fabarius: Novartis: Research Funding. Reiter:Blueprint: Consultancy, Honoraria, Other: Travel reimbursement; Deciphera: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement, Research Funding.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) &lt, 10 kDa, (II) 10–100 kDa and (III) &gt, 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10–100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample, mean ± SEM) when compared to (I) &lt, 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) &gt, 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10–100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Acute rejection is a risk factor for inferior long-term kidney transplant survival. Although T cell immunity is considered the main effector in clinical acute rejection, the role of myeloid cells is less clear. Expression of S100 calcium-binding protein A8 (S100A8) and S100A9 was evaluated in 303 biopsies before and after transplantation from 190 patients. In two independent cohorts of patients with acute rejection (n = 98 and n = 11; mostly cellular rejections), high expression of S100 calcium-binding protein A8 (S100A8) and A9 (S100A9) was related to improved graft outcome. Mechanisms of action of the S100 molecules were investigated. In the graft and peripheral blood cells, S100A8 and S100A9 expression correlated with myeloid-derived suppressor markers. In line with this finding, recombinant S100A8 and S100A9 proteins inhibited maturation and the allogeneic T cell stimulatory capacity of dendritic cells. S100A9 enhanced the production of reactive oxygen species by macrophages, which suppressed T cell activity at low concentrations in the form of hydrogen peroxide. Intragraft S100A8 and S100A9 expression linked to reduced expression of T cell immunity and tissue injury markers and higher expression of immune regulatory molecules. This study sheds new light on the importance of myeloid cell subsets in directing the outcome of T cell-mediated acute rejection. The authors find an association between levels of S100 calcium-binding proteins A8 and A9 during acute kidney transplant rejection with graft outcome, and that these myeloid cell-expressed proteins have immunoregulatory effects toward T cells.

Accumulation of DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in the pathogenesis of monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL). Here, we show increased numbers of γH2AX foci, a marker of DNA double-strand breaks (DSB), in CD19+ cells of CLL patients as compared to CD19+ cells of MBL patients and healthy individuals. Furthermore, numerous γH2AX/53BP1 foci in CLL cells suggest activation of error-prone non-homologous end-joining repair mechanisms. Signatures of DDR proteins further indicate alterations of the DDR in CLL in contrast to a largely regular activation in MBL and healthy controls. In summary, our results provide evidence for the stepwise accumulation of DNA damage in the progression of MBL towards CLL and suggest increased DNA damage, error-prone DNA repair and altered DDR signaling to be critical mechanisms of clonal evolution in MBL and CLL.

The clinical behavior of systemic mastocytosis (SM) is strongly associated with activating mutations in KIT (D816V in >80% of cases), with the severity of the phenotype influenced by additional somatic mutations, e.g. in SRSF2, ASXL1 or RUNX1. Complex molecular profiles are frequently associated with the presence of an associated hematologic neoplasm (AHN) and an unfavorable clinical outcome. However, little is known about the incidence and prognostic impact of cytogenetic aberrations. We analyzed cytogenetic and molecular characteristics of 109 patients (KIT D816V+, n=102, 94%) with indolent (ISM, n=26) and advanced SM (n=83) with (n=73, 88%) or without AHN. An aberrant karyotype was identified in SM-AHN (16/73, 22%) patients only. In patients with an aberrant karyotype additional somatic mutations were identified in 12/16 (75%) patients. Seven of 10 (70%) patients with a poor-risk karyotype, e.g. monosomy 7 or complex karyotype, and 1/6 (17%) patients with a good-risk karyotype progressed to secondary acute myeloid leukemia (n=7) or mast cell leukemia (n=1) within a median of 40 months (range 2-190, P=0.04). In advanced SM, the median overall survival (OS) of poor-risk karyotype patients was significantly shorter than in good-risk/normal karyotype patients (4 vs. 39 months; hazard ratio 11.7, 95% CI 5.0-27.3; P

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of γH2AX and 53BP1, the combined analysis of γH2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on γH2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the γH2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of γH2AX and 53BP1.

Background S100A8/A9 heterodimers are well-known alarmins that, upon release from activated or necrotic phagocytes, promote inflammation by binding to Toll-like receptor 4 (TLR4). These proteins are highly expressed in synovial phagocytes during arthritis and proved to be reliable biomarkers for monitoring disease activity in RA. Interestingly, we now identify a novel immune-regulatory mechanism of S100A8 in human monocyte-derived dendritic cells (moDCs). Objectives This study aims to analyze immune-regulatory functions of S100 proteins in human DCs. Methods MoDCs are differentiated with or without exposure to S100A8 prior to maturation with LPS. After characterization of the activation status using flow cytometry, the ability of these cells to induce autologous CD4+, CD8+, and γδ T-cell proliferation is investigated. Cytokines, secreted during development are analyzed by Luminex cytokine arrays. The metabolic state of DCs is examined by using Seahorse XFp Analyzer assays. Finally, to identify molecular mechanisms leading to an immune-regulatory phenotype, the mRNA expression of moDCs is analyzed by genome-wide gene expression arrays. Results Our results demonstrate that early exposure to S100A8 interferes with in-vitro differentiation of moDCs. Compared to controls S100A8-exposed moDCs show dramatically reduced surface expression of co-stimulatory molecules upon LPS-induced maturation. In addition, early treatment of moDCs with S100A8 alters the secretion of immune-regulatory cytokines and chemokines depending on the developmental state of moDCs. S100A8-induced effects on moDC maturation are not limited to TLR4 stimulation but rather trigger a common state of unresponsiveness. Furthermore, mitochondrial respiration and glycolytic function is diminished in S100A8-treated moDCs. As a consequence, S100A8-exposed moDCs have a reduced potential to induce autologous T-cell proliferation. We can show that these differences are mainly caused by reduced surface expression of co-stimulatory molecules on S100A8-treated moDCs. Mechanistically, genome-wide gene expression analysis reveals dramatic differences in gene expression between S100A8-exposed and conventionally differentiated moDCs. We demonstrate that S100A8 pre-treatment of moDCs significantly blocks LPS-induced gene expression during moDC activation. Interestingly, in-silico analysis of transcription factor networks predicts NFκB and C/EBPδ as master regulators of S100A8-induced effects in developing moDCs. C/EBPδ on protein level, indeed, shows reduced expression in S100A8-differentiated moDCs prior and after LPS-induced maturation when compared to conventionally differentiated moDCs. Conclusions Taken together, our results demonstrate a novel regulatory mechanism of innate immunity to prevent overwhelming immune responses. Dysregulated repression of detrimental adaptive immune responses might very well contribute to the disease phenotype in auto-immune disorders with high systemic S100A8/A9 levels. Therefore, S100A8-differentiated immune-suppressive DCs potentially represent a promising therapeutic tool to treat auto-immune diseases in the future. Disclosure of Interest None declared

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be particularly vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, as single-agents, combined with decitabine and combined with each other in CD34+ MDS/CMML cells and in CD34+ or CD34- AML cells in comparison to healthy CD34+ donor cells. The surviving fraction of CD34+ MDS/CMML cells (n = 8; 4 MDS and 4 CMML), CD34+ or CD34- AML cells (n = 18) and healthy CD34+ donor cells (n = 8) was analyzed using the CellTiter-Glo luminescent cell viability assay (Promega, Southampton, UK). Cell proliferation of untreated MDS/CMML and AML cells was determined by trypan blue exclusion assay (Merck, Darmstadt, Germany). PARP1/APE1 mRNA expression was evaluated using validated primer sets for PARP1 (Hs_PARP1_1_SG QuantiTect Primer Assay, NM_001618) and APE1 (Hs_APEX1_1_SG QuantiTect Primer Assay, ENST00000216714) (Qiagen, Hilden, Germany). Immunofluorescence microscopy of γH2AX foci was performed using a JBW301-derived mouse monoclonal anti-γH2AX antibody (Merck). Talazoparib and APE1 inhibitor III demonstrated critical anti-leukemic efficacy as single-agents in about 19-25% of MDS/CMML/AML cell samples (Figure 1A and B). Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in about 78-86% of MDS/CMML/AML cell samples. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in about 68% of MDS/CMML/AML cell samples. In summary, talazoparib and APE1 inhibitor III demonstrated substantial anti-leukemic efficacy as single-agents, in combination with decitabine and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials. Figure 1 Cytotoxic efficacy of talazoparib and APE1 inhibitor III in healthy CD34+ donor cells, in CD34+ myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) cells and in CD34+ or CD34- acute myeloid leukemia (AML) cells after 3 days of treatment. (A) The mean IC50 of talazoparib was significantly lower (*p = 0.016) in 1 MDS (MDS#2), 1 CMML (CMML#2) and 3 AML cell samples (AML#1, AML#2, AML#3) as compared to 8 healthy donor cell samples. (B) The mean IC50 of APE1 inhibitor III was substantially lower (p = 0.059) in 1 MDS (MDS#2) and 5 AML cell samples (AML#1, AML#2, AML#3, AML#6, AML#12) as compared to 8 healthy donor cell samples. Figure 1 Disclosures Fabarius: Novartis: Research Funding.

The accumulation of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in BCR/ABL1-mediated blastic transformation of chronic myeloid leukemia (CML). The aim of our study was to analyze underlying mechanisms of genetic instability with regard to DNA damage such as DNA double-strand breaks (DSB), DSB repair and DDR signaling during blastic transformation of CML. Immunofluorescence microscopy of γH2AX was performed for quantification of DSB in peripheral blood mononuclear cells (PBMC) of 8 healthy individuals, 24 chronic phase (CP)-CML patients under current/discontinued tyrosine kinase inhibitor (TKI) treatment (21 patients in deep molecular response (DMR), 3 patients in major molecular response (MMR)), 5 CP-CML patients under current/discontinued TKI treatment with loss of MMR, 3 de novo non-treated CP-CML patients and 2 blast phase (BP)-CML patients. In addition, immunofluorescence microscopy of γH2AX/53BP1 was used for semi-quantification of error-prone DSB repair. Furthermore, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was performed in PBMC of CML patients in comparison to PBMC of healthy individuals. Our analysis revealed an increase in numbers of γH2AX foci in PBMC of CP-CML patients under current/discontinued TKI treatment with loss of MMR (1.8 γH2AX foci per PBMC ± 0.4), in PBMC of de novo non-treated CP-CML patients (2.3 γH2AX foci per PBMC ± 0.7) and in PBMC of BP-CML patients (4.9 γH2AX foci per PBMC ± 0.9) as compared to the number of γH2AX foci in PBMC of healthy individuals (1.0 γH2AX foci per PBMC ± 0.1) and in PBMC of CP-CML patients under current/discontinued TKI treatment in DMR/MMR (1.0 γH2AX foci per PBMC ± 0.1) (Figure 1A and B). Analysis of co-localizing γH2AX/53BP1 foci in PBMC suggested progressive activation of error-prone nonhomologous end-joining repair mechanisms during blastic transformation in CML. Signatures of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 indicated alterations of the DDR. In summary, our data provide evidence for an accumulation of DNA damage in PBMC of CML patients towards BP-CML patients. We hypothesize that ongoing DSB generation, error-prone DSB repair and DDR alterations might be critical mechanisms of blastic transformation in CML. Figure 1 Analysis of γH2AX foci in freshly isolated peripheral blood mononuclear cells (PBMC) of healthy individuals and chronic myeloid leukemia (CML) patients. (A) Exemplary immunofluorescence microscopic images of γH2AX foci (green, Alexa 488) and cell nuclei (blue, DAPI) in PBMC of a healthy individual (HEALTHY#3), a chronic phase CML patient with a deep molecular response to tyrosine kinase inhibitor (CP-CML DMR#16), a de novo non-treated chronic phase CML patient (CP-CML#1) and a blast phase CML patient (BP-CML#2). (B) γH2AX foci levels in PBMC of healthy individuals and in PBMC of CML patients. Figure 1 Disclosures Saussele: Pfizer: Honoraria; Novartis: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Fabarius:Novartis: Research Funding.

Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrations are a hallmark of cancer. Deregulation of centriole duplication leads to supernumerary centrosomes, sister chromatid missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 45 % of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. BM cells of 34 MDS patients were examined cytogenetically. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin to analyze the centrosomal status. Umbilical cord blood specimens and BM cells of healthy persons (n = 11 and n = 4) served as controls. In addition, the protein expression of the protease separase responsible for genetic stability was examined by western blot analysis. Centrosome abnormalities were detected in 10 % (range, 4–17 %) of cells of MDS samples, but in only 2 % (range, 0–4 %) of cells of healthy controls. Normal karyotypes were found in control cells and in BM cells of 16/34 MDS patients. The incidence of centrosomal alterations was higher in BM cells of patients with cytogenetic alterations (mean, 12 %) compared to BM cells of patients without cytogenetic changes (mean, 7 %). Our results indicate that centrosome alterations are a common and early detectable feature in MDS patients and may contribute to the acquisition of chromosomal aberrations. We assume that centrosome defects could be involved in disease progression and may serve as a future prognostic marker.

Increased DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). We used immunofluorescence staining of γH2AX and 53BP1 for analyzing DNA double-strand breaks (DSB) in MDS and AML cell lines, in CD34+ selected cells of normal and MDS bone marrow (including three cases of chronic myelomonocytic leukemias) and in blasts of AML bone marrow. In addition, we screened for activation of the DDR by immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53. As compared to γH2AX foci levels in normal bone marrow samples (0.2 focus per CD34+ cell±0.0; mean±standard error of mean), increased levels of γH2AX foci were detected in 16/16 MDS bone marrow samples (2.8 foci per CD34+ cell±0.5), 18/18 AML bone marrow samples (5.5 foci per blast±0.5), 1/1 MDS cell line (6.4 foci per cell) and 6/6 AML cell lines (12.0 foci per cell±0.6). γH2AX and 53BP1 co-localized in all tested samples forming diffuse, clustered and marginal patterns. Further, DDR proteins were expressed heterogeneously suggesting impairment of the DDR. In summary, our results provide evidence for a continuous increase of DSB across the spectrum from MDS to AML in conjunction with an impaired DDR. Co-localization of γH2AX and 53BP1 indicates promotion of (in)effective nonhomologous end-joining repair mechanisms at sites of DSB. Moreover, γH2AX/53BP1 foci distribution presumably reveals a non-random spatial organization of the genome in MDS and AML.

The aim of this study was to systematically evaluate the cumulative radiation exposure and the associated lifetime-cancer-risk from diagnostic imaging in patients with Hodgkin-lymphoma-(HL) or diffuse-large-B-cell-lymphoma (DLBCL). 99 consecutive patients (53-males) diagnosed with HL or DLBCL were included in the study and followed. Based on the imaging reports, organ and effective-doses-(ED) were calculated individually for each patient and the excess lifetime risks were estimated. The average ED in the first year after diagnosis was significantly different for men (59 ± 33 mSv) and women (744 ± 33 mSv)-(p 0.05). Over all years, more than 90% of the ED resulted from CT. The average cumulative radiation risk estimated for the first year was significantly lower for men (0.76 ± 0.41%) as compared to women (1.28 ± 0.54%)-(p

High mortality rates of invasive fungal disease (IFD), especially invasive aspergillosis (IA), in immunocompromised haematological patients and current diagnostic limitations require improvement of detection of fungal pathogens by defining the optimal use of biomarkers and clinical samples. Concurrent bronchoalveolar lavage (BAL) and peripheral blood samples of 99 haematological patients with suspected IFD were investigated within a multicentre prospective study. Diagnostic performance of a galactomannan (GM) enzyme immune assay (EIA), a 1,3-β-D-glucan assay (BDG), an Aspergillus PCR, and a multifungal DNA-microarray (Chip) alone or in combination were calculated. IFD were classified as proven (n=3), probable (n=34), possible (n=33), and no IFD (n=29) according to EORTC/MSG criteria. GM, PCR, and Chip showed superior diagnostic performance in BAL than in blood, whereas specificity of BDG in BAL was poor (48% (14/29)). The combination of GM (BAL) with BDG (blood) showed sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and DOR (diagnostic odds ratio) of 92% (34/37), 93% (27/29), 94%, 90%, and 153.0, respectively. Combining GM (BAL) with PCR (BAL) showed convincing diagnostic potential for diagnosing IA with sensitivity, specificity, PPV, NPV, and DOR of 85% (17/20), 97% (28/29), 94%, 90%, and 158.7. Addition of the DNA-microarray resulted in further detection of two mucormycetes infections. In 1 out of 15 Aspergillus DNA-positive samples a triazole resistance-mediating Cyp51A mutation was found. Combination of biomarkers is superior to their sole use in diagnosing IFD, particularly IA. Integrating blood and BAL samples into a diagnostic algorithm is an advantageous approach.

Die psychogene Polydipsie mit der Folge einer ausgepragten isovolamischen Hyponatriamie wurde in der Literatur bereits mehrfach beschrieben [4, 8]. Als eine Folge dieser Elektrolytstorung kann es zu einem Hirnodem mit sekundarer Enzephalopathie bis hin zu epileptischen Anfallen kommen. Eine seltene weitere Folge stellt die Rhabdomyolyse dar mit all ihren Komplikationen (Niereninsuffizienz, Hyperkaliamie, Herzrhythmusstorungen) bis hin zum Kompartmentsyndrom bei ausgepragtem Muskelodem. Wir berichten uber einen Patienten mit einer durch eine psychogene Polydipsie induzierte Hyponatriamie, die zu einer Rhabdomyolyse mit daraus resultierendem Kompartmentsyndrom fuhrte.

High concentrations (21–44 mg kg-1 dry matter) of Mo have been identified in forage in several reclaimed mining areas in British Columbia. Since Mo concentrations greater than 5 mg kg-1 in forage dry matter may result in molybdenosis because of a secondary Cu deficiency in ruminants, a study was undertaken to determine if cattle can safely graze the reclaimed land. For 12-wk grazing periods in 1994, 1995 and 1996, 32 cow/calf pairs grazed high-Mo forage at a reclaimed mine tailings site located at the Highland Valley Copper mine near Logan lake, BC. Half of the animals in the trial received a Cu supplement (All-Trace copper bolus) and the other half served as a control group. There were no significant differences (P < 0.05) in weight gain, liver Mo, serum Cu and Mo, and milk Cu and Mo between the two treatment groups of cows. Liver Cu was higher for the Cu bolus group at certain time periods in 1994 and 1995, indicating that the bolus was effective at supplying Cu. At all times, the liver Cu levels for the animals remained above the recommended critical level of 25 mg kg-1 dry matter. Animals appeared healthy and no signs of Cu deficiency were observed. Key words: Molybdenum, molybdenosis, copper, mine tailings, reclamation, animal health

Tyrosine kinase 2 (TYK2) has a pivotal role in immunity to infection and tumor surveillance. It is associated with several cytokine receptor chains including type I interferon (IFN) receptor 1 (IFNAR1), interleukin- (IL-) 12 receptor beta 1 (IL-12Rb1) and IL-10R2. We have generated a mouse with a conditional Tyk2 null allele and proved integrity of the conditional Tyk2 locus. TYK2 was successfully removed by the use of ubiquitous and tissue-specific Cre-expressing mouse strains. Myeloid TYK2 was found to critically contribute to the defense against murine cytomegalovirus. Ubiquitous TYK2 ablation severely impaired tumor immunosurveillance, while deletion in myeloid, dendritic or T cells alone showed no effect. The conditional Tyk2 mouse strain will be instrumental to further dissect TYK2 functions in infection, inflammation and cancer.

A 2 x 3 factorial experiment was conducted to determine the effects of an implant (trenbolene acetate/estradiol or no implant) and method of estrus suppression (immunization against GnRH, melengestrol acetate, or no suppression) on growth performance and carcass characteristics of heifers fed for slaughter. At the start of a 21-d feed adaption phase, crossbred beef heifers (n = 144, 390+/-2.8 kg) were given their first dose of an anti-GnRH vaccine or started on melengestrol acetate (MGA). Thereafter, heifers were fed a high-concentrate diet (78% barley grain) for 84 d (Days 0 to 83), received implants on Day 0, a second vaccination on Day 21, and were slaughtered on Days 84 or 85. Implanting increased average daily gain (1.72 vs 1.50 kg/d, P0.01), feed efficiency (6.02 vs 6.75 kg dry matter intake/kg gain, P0.01), preslaughter weight (532 vs 513 kg, P0.01), carcass weight (301 vs 289 kg, P0.01), and ribeye area (88.6 vs 85.9 cm2, P0.05), but had no affect (P0.05) on dry matter intake, grade fat thickness, marbling score, or lean yield. Compared to heifers fed MGA, those immunized against GnRH had a greater ribeye area (90.0 vs 84.6 cm2) and lean yield (63 vs 61%), and had thinner grade fat (7.5 vs 8.6 mm; P0.05 for each). Furthermore, immunized heifers had lower (P0.001) plasma progesterone concentrations than control heifers on Days 42, 63 and 83. Heifers fed MGA had less estrus mounting activity (P0.05) and lower plasma progesterone concentrations (P0.001) than the remaining heifers. Method of estrus suppression did not affect (P0.05) preslaughter weight, average daily gain, dry matter intake, feed efficiency, carcass weight, or marbling score. In conclusion, implanting significantly increased growth performance and preslaughter and carcass weights. Compared to heifers fed MGA, immunization against GnRH significantly increased ribeye area and lean yield, and reduced grade fat thickness

The effects of sainfoin (Onobrychis viciifolia) on digestion of alfalfa (Medicago sativa) were investigated in vitro and in vivo. Fresh alfalfa and sainfoin were incubated in an artificial rumen (Rusitec) in ratios of 100:0, 75:25, 50:50, 25:75 and 0:100 (as-fed). Disappearances of dry matter and N from sainfoin were 77 and 65% of those from alfalfa, respectively. Protease and endoglucanase activities, NH3-N and methane production declined (P 0.05), but cells incorporated more 15NH3N as sainfoin in the diet increased. Chopped leaves (100:0, 95:5 and 90:10 alfalfa:sainfoin) were incubated for 48 h with diluted ruminal fluid containing 0 or 50 mg polyethylene glycol, which binds tannins. Gas and volatile fatty acid productions were similar (P > 0.05) across treatments, but including 10% sainfoin (without polyethylene glycol) reduced (P 3 concentrations between 8 and 24 h. Sainfoin tannins reduced degradation of forage protein without affecting the digestibility of the nonprotein fraction. Alfalfa herbage was fed alone or with early- to full-bloom sainfoin herbage (at 10 or 20% of ad libitum alfalfa dry matter intake) or with sainfoin hay or pellets, to eight Jersey steers in crossover trials conducted over 4 yr. Including sainfoin in the diet reduced (P

A number of observations including clinical manifestation, course, outcome, and family history, support the view that patients presenting with a major depression occurring first in late life should be treated as a nosological subgroup. In this study quantitative magnetic resonance imaging (MRI) was used to investigate volumes of different brain structures in 19 patients with late onset major depression (age of onset > 50) and 13 age matched controls. 3-D MRI sequences were acquired using a Siemens 1.5T scanner. Whole brain volume, CSF volume, volume of the frontal and temporal lobes and the volume of the amygdala-hippocampus complex were assessed using the software NMRWin. Compared to the controls, depressed patients showed a significantly lower whole brain volume and a significantly higher CSF volume, whereas volumes of the frontal and temporal lobes as well as the amygdala-hippocampus complex volumes were not significantly decreased. In addition, depressed patients exhibited a higher ventricle-brain ratio suggesting a higher degree of central atrophy compared to healthy individuals. Our results indicate that cerebral changes involving subcortical structures are of relevance in the pathogenesis of late-onset depression. Defining the aetiology of these lesions may be important for the development of preventive treatment of depression in the elderly.

A series of cells representing normal, non-tumorigenic cell lines, as well as differentiating neoplastic and undifferentiated neoplastic rat tracheal epithelial cell populations were evaluated for their ability to establish homologous and/ or heterologous cell-cell gap junction communication in culture. Gap junction communication was evaluated by flow cytometric quantitation of the transfer of the fluorescent dye calcein from a donor to a recipient cell population via gap junctions. The data indicate that normal primary cultures of rat tracheal epithelial cells, as well as non-tumorigenic cell lines and squamous cell carcinomas cell populations, retain the ability to establish both homologous and heterologous gap junction communication. In all cases an average of >48% of recipient cells had acquired calcein label during a 5-h interval of co-culture of donor and recipient cells at confluent densities. Cells harvested directly from squamous cell carcinoma tumors exhibited similar levels of cell-cell communication. In contrast, cells giving rise to undifferentiated carcinomas, as well as cells harvested from undifferentiated carcinomas, exhibited very low levels or no homologous or heterologous cell-cell communication. Cell populations exhibiting distinctly different communication phenotypes were evaluated by Northern blot analysis for expression of connexins (Cx 26, 32 and 43) and E-cadherin. Neither communicating nor non-communicating cells expressed connexin 32. Those cell populations, which established functional gap junctions, expressed E-cadherin as well as connexin 26 and/or 43. In contrast, those cell populations that lacked the ability to communicate universally lacked expression of E-cadherin, and a quarter also lacked expression of detectable levels of connexin.

The objective of this study was to determine the relative usefulness of physical examination, plain radiographs, and magnetic resonance imaging (MRI) using T1- and T2-weighted spin-echo images in evaluating the extent of urate deposition and soft tissue destruction in gouty arthritis. Seven patients with chronic tophaceous gout of the hands and wrists were examined to identify all clinically apparent tophi. Plain radiographs of the hands and wrist were obtained to further quantify soft tissue and osseous changes. MRI was then performed of the involved areas and a comparison made between soft tissue and bony changes observed by clinical examination and plain radiographs and those observed by MRI. Plain radiographs and physical examination markedly underestimate the size and extent of soft tissue and osseous involvement by tophi when compared with the findings of MRI. MRI also detects early, subclinical tophaceous deposits and indicates that urate deposits appear to spread along compartmental and fascial planes as opposed to the traditional view of strict radial growth. MRI is a useful method of determining the extent of disease in tophaceous gout and may provide information regarding the patterns of deposition and spread of monosodium urate crystals.

Systemic mastocytosis (SM) usually arises as a consequence of an acquired mutation in the receptor tyrosine kinase KIT, usually KIT D816V (>80-90% of patients). The presence of additional molecular aberrations, e.g. in SRSF2, ASXL, or RUNX1 (S/A/Rpos), has a strong adverse impact on disease phenotype and prognosis in patients with advanced SM (advSM) (Jawhar et al., Leukemia 30, 2016). However, little is known about the frequency and prognostic impact of cytogenetic aberrations and potential interactions between cytogenetic and molecular aberrations in the context of prognostication. We therefore analyzed clinical, morphological, cytogenetic and molecular characteristics of 109 patients (KIT D816V, n=102, 94%; KIT D816H, n=2, 2%; no KIT mutation, n=5, 5%) with indolent SM (ISM, n=27) and advSM (n=82) with or without an associated hematologic neoplasm (AHN) [SM-AHN, n=60; aggressive SM, n=3; mast cell leukemia (MCL), n=9; MCL-AHN, n=10)]. Next-Generation Deep Amplicon Sequencing (NGS) was performed to investigate 18 candidate genes as previously described (Schwaab et al., Blood 122, 2013). All ISM patients had a normal karyotype. In advSM, an aberrant karyotype was identified in 16/82 (18%) patients. By analogy with other myeloid neoplasms, e.g. MDS or AML, patients were classified according to their karyotype into two groups. The good-risk group (n=73) included patients with a normal karyotype (n=66) plus those with a favorable karyotype (n=7; del(5q), n=4; trisomy 8, n=1; del(1q), n=1; del(12p), n=1), while the poor-risk group (n=9) included patients with a complex karyotype (defined as ≥3 abnormalities; n=8) or monosomy 7 (n=1). Seven of 9 (78%) patients with a poor-risk karyotype and 2/7 (29%) patients with a favorable karyotype progressed to secondary AML (n=7) or MCL (n=2) within a median observation time of 13 months (range, 0-26). Overall, the median overall survival (OS) of patients with a poor-risk karyotype was significantly shorter than in patients with a good-risk karyotype (4 vs. 41 months; hazard ratio, HR, 9.6, 95% CI 3.9-23.2; P Figure Kaplan-Meier estimates of overall survival (OS) in patients with advanced systemic mastocytosis depending on the karyotype and the mutation status in the SRSF2/ASXL1/RUNX1 gene panel (S/A/R). Pairwise significantly different OS probabilities were observed for the comparison good-risk karyotype + S/A/Rneg (n=27) vs. good-risk karyotype + S/A/Rpos (n=31) vs. poor-risk karyotype (n=9). Figure. Kaplan-Meier estimates of overall survival (OS) in patients with advanced systemic mastocytosis depending on the karyotype and the mutation status in the SRSF2/ASXL1/RUNX1 gene panel (S/A/R). Pairwise significantly different OS probabilities were observed for the comparison good-risk karyotype + S/A/Rneg (n=27) vs. good-risk karyotype + S/A/Rpos (n=31) vs. poor-risk karyotype (n=9). Disclosures Valent: Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

Purpose: Increased DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). Materials and methods: We performed combined γH2AX/53BP1 immunofluorescent focus staining of DNA double-strand breaks (DSB) in MDS and AML cell lines, in CD34+ selected cells of normal and MDS bone marrow (including three cases of chronic myelomonocytic leukemias) and in blasts of AML bone marrow. In addition, we screened for activation of DDR by immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53. Results: Compared to γH2AX foci levels in normal bone marrow samples (0.18 focus per CD34+ ± 0.6), increased levels of γH2AX foci were detected in 1/1 MDS cell line (6.4 foci per cell ± 0.0), 6/6 AML cell lines (12.0 foci per cell ± 0.6), 12/12 MDS bone marrow samples (2.8 foci per CD34+ ± 0.7) as well as 12/12 AML bone marrow samples (6.0 foci per blast ± 0.6). γH2AX and 53BP1 co-localized in all tested samples forming diffuse, clustered and marginal patterns. DDR proteins were expressed heterogeneously suggesting impairment of DDR. Conclusions: Our results provide evidence for a continuous increase of constitutive DSB across the spectrum from MDS to AML in the context of an impaired DDR. γH2AX and 53BP1 co-localize in unique patterns in nuclei of MDS and AML presumably owing to a non-random spatial organization of the genome and foci formation implies promotion of ineffective nonhomologous end-joining repair mechanisms at sites of constitutive DSB. Disclosures No relevant conflicts of interest to declare.

Genetic instability due to increased DNA damage and altered DNA repair is of central significance in the initiation and progression of inherited and sporadic human leukemias. Although very rare, some inherited DNA repair insufficiency syndromes (e.g., Fanconi anemia, Bloom's syndrome) have added substantially to our understanding of crucial mechanisms of leukemogenesis in recent years. Conversely, sporadic leukemias account for the main proportion of leukemias and here DNA damaging reactive oxygen species (ROS) play a central role. Although the exact mechanisms of increased ROS production remain largely unknown and no single pathway has been detected thus far, some oncogenic proteins (e.g., the activated tyrosine kinases BCR-ABL1 and FLT3-ITD) seem to play a key role in driving genetic instability by increased ROS generation which influences the disease course (e.g., blast crisis in chronic myeloid leukemia or relapse in FLT3-ITD positive acute myeloid leukemia). Of course other mechanisms, which promote genetic instability in leukemia also exist. A newly emerging mechanism is the genome-wide alteration of epigenetic marks (e.g., hypomethylation of histone H3K79), which promotes chromosomal instability. Taken together genetic instability plays a critical role both in inherited and sporadic leukemias and emerges as a common theme in both inherited and sporadic leukemias. Beyond its theoretical impact, the analysis of genetic instability may lead the way to the development of innovative therapy strategies.

The vertebrate skeletal muscle gives rise to a series of x-ray reflexions indexed as orders (n) of 77 nm, the even orders being meridional whereas the odd orders being near-meridional. The diffraction intensities associated with these reflexions originate from the axial period of 39 nm attributable to the repeat of troponin-tropomyosin on the thin filament. In the present study, the x-ray intensities of the furthest inner reflexions, A2 (n = 2) reflexion at an axial spacing of 1/39 nm-1 and A4 (n = 4) reflexion at 1/19 nm, of this series were measured with a time resolved manner. Upon activation of the frog striated muscle, the two reflexions underwent biphasic time courses of the intensity changes. With A2 reflexion, a rapid intensity increase by 16%, being completed by the time when tension rises to 5%, was followed by a slow intensity decrease down to 50%, which was associated with the tension rise. In both phases, lateral widths remained unchanged. A4 reflexion also behaves in the same way, although the first phase (the intensity increase) was not clear due to unsatisfactory statistics. We interpret phase one as being caused by conformational change of the troponin-tropomyosin complex upon binding of Ca2+ to troponin, whereas phase two being due to direct contribution of the mass of the myosin heads bound to the thin filament, although possible contribution of conformational changes of the regulatory proteins to phase two is not excluded. The results indicated that the calcium activation of the thin filament leads the onset of the actomyosin interaction.

Reissert compounds derived from α,β-unsaturated acid chlorides were prepared. The conjugate base obtained from these Reissert compounds exhibited the following carbanion reactions: 1) Alkylation, 2) Condensation with benzaldehyde, 3) Rearrangement to give dimeric compounds rather than simple rearranged compounds. In the case of alkylated isoquinoline Reissert compounds, the attempted rearrangement led to ring annellated amines.

In the presence of trimethylsilyl trifluoromethanesulfonate, a methylene chloride solution of isoquinoline and benzoyl chloride gave N-benzoylisoquinolinium triflate (3) and N-H-isoquinolinium triflate (4). Depending on the reaction conditions, reaction of 3 may occur on the isoquinoline ring yielding a Reissert compound and 1,2-dihydroisoquinoline species. Otherwise, reactions transpire on the carbonyl of 3 to give an amide, an ester, and an anhydride.

The FIP1L1-PDGFRA fusion gene has been described in patients with eosinophilia-associated myeloproliferative disorders (Eos-MPD). Here, we report on seven FIP1L1-PDGFRA-positive patients who presented with acute myeloid leukemia (AML, n=5) or lymphoblastic T-cell non-Hodgkin-lymphoma (n=2) in conjunction with AML or Eos-MPD. All patients were male, the median age was 58 years (range, 40-66). AML patients were negative for common mutations of FLT3, NRAS, NPM1, KIT, MLL and JAK2; one patient revealed a splice mutation of RUNX1 exon 7. Patients were treated with imatinib (100 mg, n=5; 400 mg, n=2) either as monotherapy (n=2), as maintenance treatment after intensive chemotherapy (n=3) or in overt relapse 43 and 72 months, respectively, after primary diagnosis and treatment of FIP1L1-PDGFRA-positive disease (n=2). All patients are alive, disease-free and in complete hematologic and complete molecular remission after a median time of 20 months (range, 9-36) on imatinib. The median time to achievement of complete molecular remission was 6 months (range, 1-14). We conclude that all eosinophilia-associated hematological malignancies should be screened for the presence of the FIP1L1-PDGFRA fusion gene as they are excellent candidates for treatment with tyrosine kinase inhibitors even if they present with an aggressive phenotype such as AML.

Background S100A8 and S100A9, also known as myeloid-related protein-8 (MRP8) and MRP14, are widely used as biomarkers of disease activity in juvenile idiopathic arthritis. On a molecular level these proteins function as damage-associated molecular pattern molecules (DAMPs), which amplify inflammation via Toll-like receptor-4 (TLR-4) activation. Released from activated phagocytes or necrotic cells the physiologically relevant S100A8/A9 heterodimers stimulate surrounding cells in order to promote pro-inflammatory processes in innate immunity. Adaptive immunity is believed to be enhanced by these molecules via activation of dendritic cells (DCs). Interestingly, recent studies show an additional regulatory effect of S100 proteins on DCs upon prolonged exposure (Petersen et al. EMBO-J 2013). Objectives The aim of this study is to decipher the S100 protein dependent molecular mechanisms in dendritic cells which enforce aggravation or attenuation of inflammation. Methods Human monocyte derived or murine bone marrow derived DCs are differentiated with or without exposure to S100A8 for six days prior to activation with LPS. After characterization and determination of the activation status using flow cytometry, the ability of these cells to induce T-cell proliferation is investigated in an autologous, antigen-specific or allogenic fashion. To identify the molecular mechanisms leading to the observed phenotype the mRNA expression of human DCs in various stages of differentiation was screened by genome-wide gene expression arrays. Results In a model of allergic contact dermatitis S100A9 deficient mice developed elevated inflammatory responses compared to wild type mice. We could demonstrate that prolonged exposure of myeloid progenitor cells to S100 proteins blocks DC differentiation and antigen presentation resulting in a reduced OT1 and OT2 T-cell response. Human S100A8/A9 shows a similar regulatory impact on monocyte-derived DCs (moDCs), since early DC differentiation and subsequent antigen-presentation is inhibited by S100A8 as well, leading to altered T-cell responses associated with decreased proliferation and enhanced IL-10 secretion. Gene expression analysis of developing DCs indicates vast changes in prominent immune modulatory pathways like JAK/STAT, NOD, RIG-I and TLR signalling. In addition, cell adhesion and antigen presentation seems to be affected by metabolic changes (e.g. PPAR-γ regulated fatty acid biosynthesis) as well as surface expression of antigen presenting molecules like HLA and CD1c. Conclusions Taken together, our results represent a novel regulatory mechanism of innate immunity to prevent overwhelming immune responses. Disclosure of Interest None declared

The objective was to determine the effects of a recombinant fusion protein anti-GnRH vaccine on testicular development, feedlot performance, and carcass quality of beef bulls. Crossbred beef bulls (n = 58, average weight 306 kg, 9 mo of age), were randomly allocated to two groups and received either an anti-GnRH vaccine (GnRH) or placebo (Control) by intramuscular injection on d 0, 56, and 112. There were group effects (P < 0.01; as a percentage of Control) on testicular weight (53%), daily sperm production (40%), and epididymal sperm reserves (16%). There were group x time interactions (P < 0.0001) for scrotal circumference and serum testosterone concentrations; at slaughter, bulls in the GnRH group had a smaller (P < 0.05) scrotal circumference (28.3 vs 33.9 cm) and lower (P < 0.05) serum testosterone concentrations (2.2 vs 8.6 ng/mL) than those in the Control group. Average daily gain, feed intake, and feed efficiency were not different between treatments during the backgrounding phase (d 0 to 84). During the finishing phase (d 98 to 182), ADG was greater (P < 0.05) for bulls in the Control group (1.69 vs 1.42 kg/d), as was carcass weight (6.9%; P < 0.01). However, GnRH bulls had numerically better feed efficiency (6.12 vs 7.08 kg DMI/kg gain; P < 0.23) and shear force values for ribeye that were 16% lower (P < 0.14) than Control bulls, warranting further investigation. Vaccinating bulls against GnRH suppressed testicular function, with growth and carcass characteristics similar to that expected with steers.

Introduction NMDA receptor ligands have been shown to rapidly treat depression but are associated with psychotomimetic effects. GLYX-13 is an NMDA receptor glycine site functional partial agonist with ~ 25% of the agonist activity of glycine or Dserine. Animal models suggest a single intravenous dose may produce long-term efficacy without psychotomimetic effects. Objective A phase II randomized, double-blind, placebo-controlled trial was conducted to assess the efficacy of GLYX-13 with central raters. Aim To examine the effects of a single dose of GLYX-13 in subjects with inadequate response to previous treatment for MDD. Methods 48 male and 68 female subjects received a single dose of GLYX-13 (1-/5-/10-/30-mg/kg) or placebo. Central raters assessed subjects via telephone using the HDRS-17 at Screening, Baseline, Days 1, 3, 7, 14, 21 and 28. Results The a priori primary efficacy ANCOVA on pooled drug dose versus placebo was not significant for change from baseline to Day 1 on HDRS-17 total score. MMRM revealed a statistically significant reduction in HDRS-17 total score versus placebo at Day 3 for 5-mg/kg (−4.4; p Conclusion This study suggests that GLYX-13, an NMDA receptor glycine site functional partial agonist, rapidly reduces depression scores without eliciting psychotomimetic effects at therapeutic doses as assessed by central raters. Further study is indicated.

Introduction Centralized ratings by telephone have proven feasible for assessment of psychiatric diagnosis, symptom severity, and suicidality, and may be used for safety assessments in non-psychiatric trials with sites that do not employ staff experienced in psychiatric assessment. Objective To assess whether centralizing assessments with mental health experts enables immediate clinical follow-up and actionable diagnostic support for investigators. Aims To examine the feasibility of centralized ratings in a Phase III dermatology clinical trial for safety assessments. Methods 1127 subjects enrolled in a trial of medication for their dermatologic condition were assessed via telephone by central raters who administered the SCID-CT, C-SSRS and PHQ-8 at screening. At monthly visits, central raters performed the C-SSRS, PHQ-8, GAD-7 and items designed to detect emergent psychotic symptoms. Results: Screening 34 subjects were excluded on the basis of SCID-CT diagnosis. Based on diagnosis or severity, subjects were classified as being in no need of mental health services, having mild psychiatric symptoms (referred to local mental health service provider; n=33), moderate (immediate referral for psychiatric evaluation; n=17), or severe (immediate escort to emergency room; n=0). One subject reported suicidal ideation on the C-SSRS, 10 reported self-injurious behavior, and 5 reported suicidal behavior in the last year. Follow-Up No subjects reported suicidal ideation or behavior at any of the 6861 follow-up assessments. One subject reported self-injurious behavior and two reported emergent psychotic symptoms. Conclusions This study established the feasibility and acceptability of routine screening and monitoring of psychopathology and suicidality by central raters in a non-psychiatric population.

Introduction Negative syndromes in Schizophrenia are of increasing interest to drug developers. Several assessment strategies have emerged for identifying negative symptoms including the NSA-16, PANSS negative symptom subscale and the Marder subscale. Assessment of patients with schizophrenia by videoconferencing has been shown to yield results equivalent to those obtained when the scale is administered face-to-face. Objectives To assess remote assessment of negative symptoms including blinding to protocol details and visit number, effectively eliminating enrollment and expectation biases. Aims To assess whether negative symptom scales can be reliably assessed in a clinical trial by videoconferencing. Methods The PANSS and the NSA-16 were administered to 227 subjects with schizophrenia in a randomized clinical trial via live videoconferencing by 17 blinded independent central raters. Subjects were interviewed at screen, at 11 more visits over 36 weeks, and at endpoint or 1 year. On a subset of subjects, a senior clinician observed and independently rated the PANSS and NSA as a quality control measure. Results ICCs between raters and observing trainers were .98 on the NSA total score (N = 65 pairs) and .96 on the PANSS total score (N = 69 pairs). ICCs of individual NSA items ranged from .72-1.0, with a mean ICC of .91. ICCs of PANSS subscales ranged from .94 -.96 with ICCs of .95 for the Marder subscale and .94 for the negative subscale. Conclusion Excellent item-level ICCs for the NSA suggest that negative symptoms can be rated reliably by videoconferencing using well-calibrated blinded independent raters.

Introduction Inappropriate subjects may be enrolled in a study when enrollment pressures cause inflated baseline severity scores. An increasing number of studies now include methods such as blinded independent centralized ratings (CR) to ensure that appropriate subjects are entered into the trial. Post-baseline factors such as functional unblinding, expectation bias and rater drift can also affect outcomes. Objectives Independent raters, blind to study visit, can minimize functional unblinding and expectation bias. Continuous calibration of CR can minimize rater drift. Aims To examine studies with both site ratings (SR) and CR to determine how critical post-baseline blinding and continuous calibration are. Methods A trial of acute schizophrenia used CR for the PANSS and SR for the BPRS on the same subjects. A Parkinson's psychosis study used CR in the US and SR ex-US to assess subjects using the SAPS. A GAD trial used CR of subjects enrolled by SRs’ SIGH-A evaluations. Results In the schizophrenia trial, CR separated the active comparator and one of two test arms. SR separated the active comparator but neither test arm. In the Parkinson's psychosis study, pimavanserin showed greater separation with CR than SR. In the GAD trial, CR had lower placebo response than SR, independent of subject selection. Conclusions Data from several studies support the continued importance of rater blinding and independence, post subject selection. Results suggest that precision of ratings beyond baseline can increase the sensitivity of findings in a clinical trial, decrease placebo response rates and potentially eliminate Type II errors.