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We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity in monocyte/macrophage models and induces ERK signalling. In the present study we investigated whether MBZ induced ERK activation is shared by other tubulin binding agents (TBAs) and if it is observable also in other human cell types. Curated gene signatures for a panel of TBAs in the LINCS Connectivity Map (CMap) database showed a unique strong negative correlation of MBZ with MEK/ERK inhibitors indicating ERK activation also in non-haematological cell lines. L1000 gene expression signatures for MBZ treated THP-1 monocytes also connected negatively to MEK inhibitors. MEK/ERK phosphoprotein activity testing of a number of TBAs showed that only MBZ increased the activity in both THP-1 monocytes and PMA differentiated macrophages. Distal effects on ERK phosphorylation of the substrate P90RSK and release of IL1B followed the same pattern. The effect of MBZ on MEK/ERK phosphorylation was inhibited by RAF/MEK/ERK inhibitors in THP-1 models, CD3/IL2 stimulated PBMCs and a MAPK reporter HEK-293 cell line. MBZ was also shown to increase ERK activity in CD4+ T-cells from lupus patients with known defective ERK signalling. Given these mechanistic features MBZ is suggested suitable for treatment of diseases characterized by defective ERK signalling, notably difficult to treat autoimmune diseases.

Objectives/Hypothesis: Postoperative tinnitus and taste disturbances after myringoplasty are more common than previously reported.Study Design: This study was a retrospective analysis of prospectiv ...

Data from patients registered for myringoplasty during 2002 to 2012 in the Swedish National Quality Registry for Myringoplasty.Both conventional myringoplasty and fat-graft techniques were used aimed at healing the tympanic membrane in noninfected ears.Analysis was performed on data in a national database collected from 32 ear, nose, and throat clinics. Surgical procedures and outcomes, and patient satisfaction from a questionnaire were studied.The database was comprised of 3,775 surgical procedures, with follow-up available for analysis. One-third were children under the age of 15 years. The most common indication for surgery was infection prophylaxis. The overall healing rate of the tympanic membrane after surgery was 88.5%, with a high mean patient satisfaction. Complications registered were postoperative infection, tinnitus, or taste disturbance that occurred in 5.8% of patients.Swedish results for a large number of patients who completed myringoplasty are presented. The success rate in this study is comparable to other studies, and good patient-reported outcome measures of myringoplasty are presented. Databases for surgical procedures and clinical audits are systematic processes for continuous learning in healthcare. This study shows that clinical databases can be utilized to analyze national results of surgical procedures.2b Laryngoscope, 127:2389-2395, 2017.

To present hearing results after successful primary myringoplasty surgeries registered in the Swedish Quality Registry for Myringoplasty and to evaluate the chance of hearing improvement and the risk of hearing loss.A retrospective nationwide cohort study based on prospectively collected registry data between 2002 and 2012.Registry data from secondary and tertiary hospitals performing myringoplasty.Patients with healed tympanic membrane after primary myringoplasty surgery performed from 2002 to 2012 in Sweden.Postoperative hearing results, hearing gain and air-bone gap (ABG).In 2226 myringoplasties, air conduction audiograms were recorded, and the average preoperative pure tone average (PTAHearing results after successful myringoplasty surgery are often favourable, but although the tympanic membrane is healed, hearing improvement is not guaranteed, and hearing deterioration can also occur.

Highlights • Fifteen cytokines were increased at ICU admission in Covid-19 patients. • A relatively moderate increase in cytokine concentrations was observed. • IL-1ra, IL-6 and IP-10 correlated with respiratory failure and acute kidney injury. • IL-8 may be a future biomarker due to its correlation with 30-day mortality., Background The infection caused by SARS CoV-2 has been postulated to induce a cytokine storm syndrome that results in organ failure and even death in a considerable number of patients. However, the inflammatory response in Corona virus disease-19 (Covid-19) and its potential to cause collateral organ damage has not been fully elucidated to date. This study aims to characterize the acute cytokine response in a cohort of critically ill Covid-19 patients. Method 24 adults with PCR-confirmed Covid-19 were included at time of admission to intensive care a median of eleven days after initial symptoms. Eleven adult patients admitted for elective abdominal surgery with preoperative plasma samples served as controls. All patients were included after informed consent was obtained. 27 cytokines were quantified in plasma. The expression of inflammatory mediators was then related to routine inflammatory markers, SAPS3, SOFA score, organ failure and 30-day mortality. Results A general increase in cytokine expression was observed in all Covid-19 patients. A strong correlation between respiratory failure and IL-1ra, IL-4, IL-6, IL-8 and IP-10 expression was observed. Acute kidney injury development correlated well with increased levels of IL-1ra, IL-6, IL-8, IL-17a, IP-10 and MCP-1. Generally, the cohort demonstrated weaker correlations between cytokine expression and 30-day mortality out of which IL-8 showed the strongest signal in terms of mortality. Conclusion The present study found that respiratory failure, acute kidney injury and 30-day mortality in critically ill Covid-19 patients are associated with moderate increases of a broad range of inflammatory mediators at time of admission.

Background Melflufen (melphalan flufenamide) is a first-in-class peptide-drug conjugate (PDC) that targets aminopeptidases and rapidly releases alkylating agents into tumor cells. Osteolytic lesions are a hallmark of multiple myeloma and occur in almost all MM patients, causing severe bone pain and pathological fractures. Previous reports indicate that melphalan enhances osteoclastogenesis. On other other hand, melflufen is able to suppress proliferation of malignant myeloid cells including osteoclast precursors, monocytes. In the present study we therefore evaluated the direct effects of melflufen and melphalan on human osteoclastogenesis using cultured peripheral blood CD14+ monocytes. Methods CD14+ osteoclast precursors were isolated from peripheral blood. Osteoclastogenesis was stimulated by adding RANKL and M-CSF to CD14+ osteoclast precursors and assessed by tartrate-resistant acid phosphatase (TRAP) staining using para-nitrophenyl phosphate (PNPP). Osteoclastic activity was assessed by two assays. First, TRAP activity was measured by quantification of resorption pits on osteo assay synthetic surface made of crystalline calcium phosphate coating mimicking living bone material. Second, collagen degradation was quantified by release of C-terminal telopeptide of type I collagen (CTx) from mineralized bone slices. The proliferation of mature osteoclasts and their precursors in the presence of melflufen and melphalan was assessed using cytotoxicity assays. RNA isolated from osteoclast precursors and mature osteoclasts was subject to gene expression analysis by microarray. Results Melflufen inhibited RANKL-induced osteoclast formation at 1 µM concentration, while 25 µM melphalan was needed to initiate osteoclastogenesis inhibition. However, only extremely high concentrations of melflufen (10 µM) and melphalan (100 µM) affected mature osteoclasts and their TRAP activity. Cytotoxicity assays have demonstrated that melflufen, but not melphalan, can inhibit proliferation of monocytes, osteoclast progenitor cells, at clinically relevant concentrations. When added during osteoclastogenesis, melflufen has significantly inhibited TRAP phosphatase activity on crystalline calcium phosphate-coated surface at 1 µM, whereas 25 µM of melphalan was required to achieve the same effect. As for collagen degradation, the addition of 1 µM melflufen completely suppressed the CTx fragment release. In a parallel assay, 10 µM of melphalan was necessary to produce the same effect. Noteworthy, we did not observe any positive effect of melphalan on osteoclastogenesis or proliferation of osteoclasts. Gene expression analysis showed that mature osteoclasts express higher levels of bone lytic genes ACP5/TRAP, CTSK, MMP2, MMP7, and MMP9. In addition, CD276 and PDCD1LG2 mRNAs encoding B7 family immune checkpoint molecules B7-H3 and PD-L2 are also strongly up-regulated upon osteoclastogenesis. Discussion Myeloma patients often suffer from severe bone pain associated with osteolytic bone lesions. These lesions are the result of excessive osteoclastogenesis induced by multiple myeloma in the bone marrow microenvironment. Moreover, this process creates an immunosuppressive microenvironment since osteoclasts express immune checkpoint molecules known to negatively control activity of T lymphocytes (PD-L2, B7-H3) and NK cells (B7-H3). In this study, we have shown that a novel anti-myeloma PDC melflufen efficiently inhibits osteoclastogenesis and associated collagen degradation and bone resorption when used at clinically relevant concentrations. Melphalan could also induce a reduction of bone resorption and collagen release, however high and clinically irrelevant concentrations were necessary to obtain these effects. Given these results, it is plausible that melflufen-treated MM patients might achieve a reduction in bone pain, and this should be assessed in more detail in the follow-up studies. Also, elimination of immunosuppressive osteoclasts by melflufen may provide a synergistic effect between melflufen and immunotherapy while treating MM patients. This hypothesis is currently being tested in clinical trials. Disclosures Byrgazov: Oncopeptides AB: Current Employment. Gullbo:Theradex: Current Employment; Oncopeptides AB: Consultancy, Current Employment, Current equity holder in publicly-traded company. Slipicevic:Oncopeptides AB: Current Employment. Lehmann:Oncopeptides AB: Current Employment, Current equity holder in publicly-traded company. Larsson:Oncopeptides AB: Current equity holder in publicly-traded company. Fryknäs:Oncopeptides AB: Research Funding.

Mebendazole (MBZ), a drug commonly used for helminitic infections, has recently gained substantial attention as a repositioning candidate for cancer treatment. However, the mechanism of action behind its anticancer activity remains unclear. To address this problem, we took advantage of the curated MBZ-induced gene expression signatures in the LINCS Connectivity Map (CMap) database. The analysis revealed strong negative correlation with MEK/ERK1/2 inhibitors. Moreover, several of the most upregulated genes in response to MBZ exposure were related to monocyte/macrophage activation. The MBZ-induced gene expression signature in the promyeloblastic HL-60 cell line was strongly enriched in genes involved in monocyte/macrophage pro-inflammatory (M1) activation. This was subsequently validated using MBZ-treated THP-1 monocytoid cells that demonstrated gene expression, surface markers and cytokine release characteristic of the M1 phenotype. At high concentrations MBZ substantially induced the release of IL-1β and this was further potentiated by lipopolysaccharide (LPS). At low MBZ concentrations, cotreatment with LPS was required for MBZ-stimulated IL-1β secretion to occur. Furthermore, we show that the activation of protein kinase C, ERK1/2 and NF-kappaB were required for MBZ-induced IL-1β release. MBZ-induced IL-1β release was found to be dependent on NLRP3 inflammasome activation and to involve TLR8 stimulation. Finally, MBZ induced tumor-suppressive effects in a coculture model with differentiated THP-1 macrophages and HT29 colon cancer cells. In summary, we report that MBZ induced a pro-inflammatory (M1) phenotype of monocytoid cells, which may, at least partly, explain MBZ's anticancer activity observed in animal tumor models and in the clinic.

Intratumoral heterogeneity is a hallmark of glioblastoma multiforme and thought to negatively affect treatment efficacy. Here, we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability among clones, including a range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-mesenchymal shift in the transcriptome. Multitherapy resistance was associated with a semi-stable cell state that was characterized by an altered DNA methylation pattern at promoter regions of mesenchymal master regulators and enhancers. The gradient of cell states within the GIC compartment constitutes a distinct form of heterogeneity. Our findings may open an avenue toward the development of new therapeutic rationales designed to reverse resistant cell states.

Glioblastoma multiforme (GBM) remains in the group of incurable malignancies, with a median survival of 15 months. Intratumoral heterogeneity is a key factor driving relapse by providing the basis for escape of therapy-resistant cells. While the heterogeneous genomic and transcriptomic landscape of cancers is currently thoroughly characterized, phenotypic heterogeneity within tumors is less well understood. The overall goal in this study was to identify pre-existing treatment resistant clones with disease relapse potential and biomarkers linked to resistance vs sensitivity. To enable functional studies of isolated tumor clones we adopted an in vitro subcloning strategy. An adherent glioma neural stem cell culturing protocol was used to enrich for cells with stem-like properties and to permit efficient phenotypic screening. We established six libraries of clonal cell cultures from fresh GBM surgical specimens, corresponding to five different patient tumors. In all 708 clonal cultures were expanded. These were considered representatives of the self-renewing compartment within the tumor cell population. We found a remarkable variation in drug and radiation response within the clone libraries. A striking observation was that clones resistant to one drug, also tended to be resistant to most of the drugs we used, regardless of the drug's mechanism of action. This indicates that resistance in large parts is mediated by a general mechanism. Most clones carried genetic aberrations that were unique or found only in a few clones but it was primarily the transcriptome data that demonstrated a clear link to the phenotypic data. A continuous gradient between multi-resistance and sensitivity was found to be connected to a gradual transition between a mesenchymal (MES), in resistant clones, and a more proneural (PN) and proliferative character in sensitive clones. The continuous distributions and lack of discrete groups in clonal response phenotypes and in linked signatures points to a general cell-state related resistance mechanism. We also found that resistant clones had a lower methylation level in promoters of known master regulators of MES subtype associated genes. This indicates that the phenotypic heterogeneity is driven by fluctuations in the epigenetic status. Taken together, our findings imply that intratumoral heterogeneity in GBM includes general clonal resistance mechanisms among glioma- initiating cells driven by epigenetic mechanisms. This evokes hope that new therapeutic approaches involving epigenetic reprogramming can be applied to sensitize cells toward conventional treatment. Citation Format: Anna Segerman, Maria Niklasson, Caroline Haglund, Tobias Bergstrom, Malin Jarvius, Yuan Xie, Demet Caglayan Simov, Annika Hermansson, Marianne Kastemar, Zeinab Naimaie-Ali, Malin Berglund, Ann Westermark, Magnus Sundstrom, Goran Hesselager, Lene Uhrbom, Mats Gustafsson, Rolf Larsson, Marten Fryknas, Bo Segerman, Bengt Westermark. GBM exhibits phenotypic microheterogeneity and harbors pre-existing multi- resistant clones with a mesenchymal transition signature. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2415.