Extended Survival Time Research Articles

Knockdown of milk-fat globule EGF factor-8 suppresses glioma progression in GL261 glioma cells by repressing microglial M2 polarization.

Tumor-associated microglial cells promote glioma growth, invasion, and chemoresistance by releasing inflammatory factors. Milk fat globule EGF factor 8 protein (MFG-E8), a secreted glycoprotein, is closely related to tissue homeostasis and anti-inflammation. In the present study, we investigated the role of MFG-E8 in microglial polarization and glioma progression in vitro and in vivo. We found that glioma cells secrete comparable amounts of MFG-E8 in culture media to astrocytes. Recombinant MFG-E8 triggered microglia to express the M2 polarization markers, such as arginase-1 (ARG-1), macrophage galactose-type C-type lectin-2 (MGL-2), and macrophage mannose receptor (CD206). Forced expression of MFG-E8 in BV-2 microglia cells not only promoted IL-4-induced M2 polarization but also inhibited lipopolysaccharide (LPS)-induced M1 microglial polarization. Mechanistic studies demonstrated that recombinant MFG-E8 markedly induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the STAT3 inhibitor stattic significantly blocked MFG-E8-induced ARG-1 expression. Administration of antibody against MFG-E8 and knockdown of its receptor, integrin β3, significantly attenuated MFG-E8-induced ARG-1 expression. Similarly, knockdown of MFG-E8 also markedly reduced IL-4-induced M2 marker expression and increased LPS-induced M1 marker expression in microglia cells. Moreover, the knockdown of MFG-E8 in GL261 glioma cells inhibited cell proliferation and enhanced chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), which was likely associated with the downregulation of FAK/AKT activation and STAT3/cyclin D1 signaling. The murine GL261 glioma experimental model demonstrated that knockdown of MFG-E8 significantly reduced tumor size and extended survival times. Additionally, attenuated CD11b+ cell infiltration and reduced CD206+ expression in CD11b+ cells were also observed in an MFG-E8 knockdown GL261 murine glioma model. These results suggested that inhibition of MFG-E8 might hamper the immunosuppressive microenvironment in gliomas and therefore ameliorate tumor progression.

Albumin-Based Nanotheranostic Probe with Hypoxia Alleviating Potentiates Synchronous Multimodal Imaging and Phototherapy for Glioma.

Highly infiltrative and invasive glioma cells obscure the boundary between tumor and normal brain tissue, making it extremely difficult to precisely diagnose and completely remove. The combination of multimodal imaging with effective treatments to diagnose precisely and guide surgery and therapy accurately is desperately needed for glioma in the brain. Here, we report a biomimetic catalase-integrated-albumin phototheranostic nanoprobe (ICG/AuNR@BCNP) to realize multimodal imaging, amplify phototherapy, and guide surgery for glioma after penetrating the blood-brain barrier, accumulating into deep-seated glioma via albumin-binding protein mediated transportation. The phototheranostic nanoprobe enabled fluorescence, photoacoustic, and infrared thermal imaging with desirable detecting depth and high signal-to-background ratio for clearly differentiating brain tumors from surrounding tissues. Meanwhile, the nanoprobe could effectively induce local hyperthermia and promote the level of singlet oxygen based on alleviated hypoxic glioma microenvironment by decomposing endogenous hydrogen peroxide to oxygen to amplify phototherapy. Thus, significant inhibition of glioma growth, extended survival time, alleviated tumor hypoxia, improved apoptosis, and antiangiogenesis effects were exhibited in several animal models including the periphery and the brain through intravenous or intratumoral injection, meanwhile with low toxicity to normal tissue. The phototherapy was also guided by the assistance of external bioluminescence, magnetic resonance, and positron emission tomography imaging. Moreover, the nanoprobe could accurately guide the glioma resection. These results suggest that the phototheranostic nanoprobe is a promising nanoplatform specifically for glioma to achieve multimodal diagnosis, effective phototherapy, and accurate imaging-guided surgery.

Cardiorespiratory fitness and cancer in men with cardiovascular disease: Analysis from the Veterans Exercise Testing Study.

Cardiovascular disease and cancer share similar risk factors and are the leading causes of death worldwide. This study aimed to assess the association between cardiorespiratory fitness, cancer incidence and cancer mortality in men with cardiovascular disease. Baseline cardiorespiratory fitness (treadmill exercise test) was assessed in 565 men aged 58.9 ± 17 with documented cardiovascular disease and free from any malignancy. Cox multivariable hazard models, population attributable fraction and exposure impact number were analyzed in model accounting for competing events for cancer outcomes. Mean cardiorespiratory fitness was 7.6 ± 3.4 metabolic equivalents. During a 12.0 ± 7.5 year follow-up, 147 participants developed any type of cancer, 70 died from cancer, and five died from causes other than cancer as competing events. Compared to low cardiorespiratory fitness (<5 metabolic equivalents), moderate (5-10 metabolic equivalents) and high cardiorespiratory fitness (>10 metabolic equivalents) were associated with 50% (0.50, 95% confidence interval (0.27-0.91)) and 68% (0.32 (0.11-0.88)) reduced risks for cancer mortality (p trend = 0.026), respectively. Survival time was longer among individuals with moderate (20.8 (19.7-22) years) and high (24.9 (23-26.7) years) compared to low cardiorespiratory fitness (17.2 (15.1-19.3) years), p < 0.001. Population attributable fraction and exposure impact number for cancer mortality were 13% (4.1-17.7) and 10.8 (5.1-56.4), p = 0.01, respectively. Cardiorespiratory fitness was not associated with cancer incidence. Higher cardiorespiratory fitness was independently associated with lower risk of cancer mortality and extended survival time in men with cardiovascular disease, although it was not associated with cancer incidence. Improving cardiorespiratory fitness through supervised exercise rehabilitation programs could potentially serve as a cost-effective public-health strategy for secondary prevention and survivorship in men with cardiovascular disease.

Competitive interactions between entomopathogenic nematodes and parasitoid venom

AbstractEntomopathogenic nematodes and parasitoid larvae of some wasps play important roles in the natural control of the pest insects. However, it has not been excluded that competition between nematodes and wasps may in some cases reduce their efficacy in the pest control. Using caterpillars of Spodoptera littoralis, we examined interactions between the nematode Steinernema carpocapsae and the venom of the parasitoid Habrobracon hebetor. The survival of S. littoralis caterpillars was reduced in a dose‐dependent manner when 5 to 500 nematodes or 0.005–0.1 venom units were applied to single caterpillars. High doses of either nematodes or the venom caused death within 1–3 days in all treated hosts. The low doses of nematodes killed caterpillars within a week, in some cases when they attempted to pupate. Caterpillars receiving low venom doses were characterized by extended survival time terminated with death due to starvation. Combined treatment of nematodes and the venom were mutually synergistic and elicited severe lethal effects. The nematodes were fully resistant to the venom and can feed and grow on the symbiotic bacteria in vitro. The venom impairs food processing and causes death of caterpillars due to starvation. Disruption of the hormonal regulation of metamorphosis by ecdysteroids and juvenile hormone could be responsible for defective moults block at different stages of the moulting process, regionally restricted moulting, moults to “intermediates” combining regions of newly secreted larval and pupal cuticles.

Accomplishing an adaptive clinical trial for cancer: Valuation practices and care work across the laboratory and the clinic.

A new generation of adaptive, multi-arm clinical trials has been developed in cancer research including those offering experimental treatments to patients based on the genomic analysis of their cancer. Depending on the molecular changes found in patients’ cancer cells, it is anticipated that targeted and personalised therapies will be made available for those who have reached the end of standard treatment options, potentially extending survival time. Results from these trials are also expected to advance genomic knowledge for patients in the future. Drawing on data from a qualitative study of one such trial in the UK, comprising observations of out-patient clinic appointments, out-patient biopsy procedures, laboratory work, and interviews with practitioners, this paper explores how the clinical and research value of one such trial was accomplished in everyday practice by focussing on the work of clinical trials and laboratory staff across recruitment, laboratory analysis, and results management. In the face of numerous potential set-backs, disappointments and failure, we explore how practitioners worked to balance the need to meet established measures of value such as numbers of patients recruited into the trial, alongside cultivating the value of positive affects for patients by managing their expectations and emotions. This care work was performed primarily by practitioners whose roles have historically been devalued in healthcare practice and yet, as we show, were critical to this process. We conclude by arguing that as complex multi-arm adaptive trials become more commonplace, we need to attend to, and render visible, the dynamic and care-full valuation practices of backstage practitioners through which experimental biomedicine is accomplished, and in doing so show that care both achieves clinical and research value, and is also a series of practices and processes that tends to tissue, patients and staff in the context of ever-present possibility of failure.

Growth and Development of Helicoverpa armigera (Lepidoptera: Noctuidae) Larvae Infected by Heliothis virescens ascovirus 3i (HvAV-3i).

Although the cotton bollworm Helicoverpa armigera has traditionally been controlled by application of chemical pesticides, chemical control selects for resistance, pollutes the environment, and endangers human health. New methods for controlling H. armigera are therefore needed. Heliothis virescens ascovirus 3i (HvAV-3i) is a recently identified virus of the lepidopteran larvae. We tested the effects of HvAV-3i on H. armigera larvae following oral ingestion of HvAV-3i-containing hemolymph (about 1.0 × 1010 virus genome copies per larvae) and following injection of HvAV-3i-containing hemolymph by insertion of a needle. Following oral ingestion, first-instar to fifth-instar larvae grew and developed normally. Following needle injection, in contrast, the corrected mortality of third and fourth instars was 88.9 ± 2.1 and 93.7 ± 3.4%, respectively. Food intake was significantly lower for larvae injected with virus-containing hemolymph than with virus-free hemolymph. Larvae injected with virus-containing hemolymph had extended survival times and could not complete the pre-pupal stage. These results indicate that inoculation of HvAV-3i via needle injection, but not via oral ingestion, significantly reduced the growth and development of H. armigera larvae.

Abstract OT3-13-01: Estimation of willingness-to-pay for breast cancer treatments through contingent valuation method in Japanese breast cancer patients (JCOG1709A)

Abstract Background: Medical expenses are on an increasing trend due to "medical innovation". The Japanese government introduced health technology assessment (HTA) in April 2016. HTA is the systematic evaluation process of scientific value, economic, social and ethical issues related to medical technology with fair and robust methods while ensuring transparency. Two achievements are expected in our research. First, we establish the evaluation standard for the interpretation of the results of cost-effectiveness analysis (CEA) of clinical trials for a standard treatment for breast cancer. Next, we can re-evaluate the HTA's decision making based on the viewpoint of general Japanese population from the perspective of cancer patients. We believe this research can contribute to the creation of a healthcare system that is meaningful to patients, clinicians, industries, and healthcare policymakers. Specific Aims: The aim of this study is to prove how Japanese breast cancer patients who received treatment in Japan consider the financial value (willingness to pay; WTP) for their life and health by using the Contingent Valuation Method (CVM), prospectively. Based on our results, we will construct an evaluation axis from patients’ viewpoint for the cost effectiveness of clinical trials to establish standard treatments for breast cancer. Key Eligibility Criteria: For Japanese breast cancer patients aged 20 to 79 who meet the followings. Unilateral breast cancer patients who fulfill either i) or ii) below i) Primary breast cancer of clinical stage 0 through III for which surgery and/ or chemotherapy and / or radiotherapy has been completed as initial treatment and that is currently undergoing regular follow-up observations or postoperative endocrine therapy. ii) Undergoing endocrine therapy or first-line through third-line chemotherapy for metastatic or recurrent cancer at least 2 months. Trial Design: This study will be conducted by the Breast Cancer Study Group of the Japan Clinical Oncology Group (JCOG). We will investigate WTP for hypothetical scenarios for primary breast cancer patients (PBC) and metastatic breast cancer patients (MBC), using a two-step dichotomous-choice method in CVM. For PBC, we will evaluate the monetary value of the prevention of recurrent cancer. Therefore, to investigate WTP, we will present hypothetical new treatments for preventing breast cancer death to these patients. In MBC, new treatments for extending survival time is presented as hypothetical scenarios and the patients’ WTP is investigated. Statistical Methods: Questionnaire data that is returned by 4 weeks (postmark deadline) after enrollment is completed at the facility and will be considered valid and analyzed. While the WTP amounts of individual responders are not clarified with the two-step dichotomous-choice method, analyses will proceed considering the obtained responses as follows. With the two-step dichotomous-choice method, if the first stage response is yes, a higher amount will be displayed for the second stage. If the first stage response is no, a lower amount will be shown for the second stage. Target Accrual: The study will be conducted in two stages. First, 168 patients (84 patients with PBC and 84 patients with MBC) will be examined to determine their WTP for setting up the dichotomous-choice method survey form. Next, 1,596 patients (798 patients with PBC and 798 patients with MBC) will be surveyed on their WTP for hypothetical scenarios in CVM. Approximately 1800 patients with primary breast cancer and stable metastatic breast cancer are enrolled. Contact information for people with a specific interest in the trial: Tsuguo Iwatani, MD, PhD. Department of Breast Surgery, National Cancer Center Hospital East tiwatani@east.ncc.go.jp Citation Format: Tsuguo Iwatani, Fumikata Hara, Hiroji Iwata, Tadahiko Shien, Takashi Hojo, Yasuaki Sagara, Norikazu Masuda, Masato Takahashi, Shigehira Saji, Tomomi Fujisawa, Naoto Kondo, Akihiko Shimomura. Estimation of willingness-to-pay for breast cancer treatments through contingent valuation method in Japanese breast cancer patients (JCOG1709A) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-13-01.

Development of an IL-17A DNA Vaccine to Treat Systemic Lupus Erythematosus in Mice.

The interleukin-17 (IL-17) family, especially IL-17A, plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). This study developed an IL-17A epitope vaccine to treat SLE in NZBWF1 and MRL/lpr mouse models. A plasmid vector encoding a hepatitis B core (HBc)-IL-17A epitope fusion protein was injected using electroporation into the skeletal muscle of NZBWF1(New Zealand Black mice x New Zealand White mice F1 hybrid strain) or MRL/lpr mice three times at 2-week intervals. As a result, anti-IL-17A antibodies were successfully produced in the HBc-IL-17A group. Accordingly, serum tumor necrosis factor alpha (TNF-α) concentrations were significantly reduced in the HBc-IL-17A group. According to pathological analysis, the IL-17A DNA vaccine significantly suppressed renal tissue damage and macrophage infiltration. Consequently, the survival rate was significantly improved in the HBc-IL-17A group. In addition, we evaluated the antigen reactivity of splenocytes from IL-17A-immunized mice using an enzyme-linked immune absorbent spot (ELISPot) assay for safety evaluation. Splenocytes from IL-17A-immunized mice were significantly stimulated by the HBc epitope peptide, but not by the IL-17A epitope or recombinant IL-17A. These results indicate that the IL-17A vaccine did not induce autoreactive T cells against endogenous IL-17A. This study demonstrates for the first time that an IL-17A DNA vaccine significantly reduced organ damage and extended survival time in lupus-prone mice.

Effect of Formulated Artocarpus champeden Extract on Parasite Growth and Immune Response of Plasmodium berghei-Infected Mice.

Background The ethanol extract of Artocarpus champeden stem bark (ACEE) has been proven to exhibit antimalarial activity. Despite the antimalarial effects observed, mechanisms of immune response to explain the antimalarial activity of ACEE remain poorly characterized. Here, we show the production of pro- and anti-inflammatory cytokines T helper 1 (Th1: IFN-γ, TNF-α) and T helper 2 (Th2: IL-10) from Plasmodium berghei-infected mice treated with formulated ACEE in order to better characterize the mechanism behind ACEE's antimalarial activity. In addition, we have also determined the effect of formulated ACEE on parasite growth and liver function. Methods Balb/c mice were infected with P. berghei strain ANKA and then administered daily doses of ACEE at a dose of 20, 50, and 100 mg/kg BW, and survival time was recorded. We determined the presence of P. berghei strain ANKA and then administered daily doses of ACEE at a dose of 20, 50, and 100 mg/kg BW, and survival time was recorded. We determined the presence of P. berghei strain ANKA and then administered daily doses of ACEE at a dose of 20, 50, and 100 mg/kg BW, and survival time was recorded. We determined the presence of γ, TNF-α) and T helper 2 (Th2: IL-10) from Results We found that formulated ACEE inhibited parasite growth and showed the highest antimalarial activity at 100 mg/kg BW. AST and ALT levels were found to be in the normal range, and there was no significant difference among control and treatment groups (P > 0.05). Infected mice treated with formulated ACEE showed a significant increase in the production of IFN-γ, TNF-α) and T helper 2 (Th2: IL-10) from Conclusion This study suggests that the administration of ACEE was effective in inhibiting P. berghei strain ANKA and then administered daily doses of ACEE at a dose of 20, 50, and 100 mg/kg BW, and survival time was recorded. We determined the presence of γ, TNF-α) and T helper 2 (Th2: IL-10) from

EXTH-45. THERAPEUTIC EFFICACY OF MUTANT ISOCITRATE DEHYDROGENASE 1 (IDH1) INHIBITOR SYC-435 WITH STANDARD THERAPY IN PATIENT-DERIVED IDH1 MUTANT GLIOMA XENOGRAFT MOUSE MODELS

Abstract Mutation in isocitrate dehydrogenase 1 (IDH1) occurs in &gt;70% of WHO grade II/III astrocytomas, oligodendrogliomas and secondary glioblastoma. The mutant enzyme catalyzes the reduction of α-ketoglutaric acid to D-2-hydroxyglutaric acid, leading to cancer initiation. In this study, we examined therapeutic efficacy of SYC-435 (1-hydroxypyridin-2-one), a newly developed mutant IDH1inhibitor, in IDH1 mutant gliomas. IDH1 R132H mutation (homozygous) was detected in BT142 anaplastic oligoastrocytoma (AOA) and R132C mutation (mutant allele frequency 39–50%) in V0914AOA by pyrosequencing. Suppression of cell growth by SYC-435 was observed with more sensitive of mutant over wild-type IDH1. Patient-derived orthotopic xenograft mouse models of BT142AOA and V0914AOA were treated with vehicle, SYC-435 (15 mg/kg/day x 28 days), temozolomide (50 mg/kg/day x 5 days)/fractionated radiation (2 Gy/day x 5 days) (standard therapy), and SYC-435/standard therapy starting 2 weeks after intracranial tumor implantation. Log rank analysis showed SYC-435 alone did not alter survival times. Although standard therapy significantly prolonged survival times in both models (P&lt; 0.0005), SYC-435/standard therapy further extended survival times (P&lt; 0.05) in V0914AOA and exhibited similar trend in BT142AOA. Elevation of 2-HG/α-KG ratio and methylation of H3K4/H3K9 in V0914AOA tumor compared to wild-type model was detected at the end of treatments. SYC-435 with/without standard therapy tended to reduce 2-HG/α-KG ratio and dramatically reduced methylation of H3K4/H3K9. RNA-seq analysis showed sirtuin signaling pathway, mitochondrial dysfunction and oxidative phosphorylation pathways with mitochondrial DNA (mtDNA) encoded molecules were highly affected by all treatments. However, mtDNA regulation did not correlate to survival benefits. In conclusion, SYC-435 possesses anti-tumor effects that are more sensitive in IDH1 mutant gliomas and generated strong synergistic activities with standard therapies for survival benefits with reduced methylation of H3K4/H3K9. Treatments significantly affected mtDNA but significance to survival benefits remains to be elucidated. Our data support the clinical testing of SYC-435 in patients with IDH1 mutant glioma.

Minimal Residual Disease Status as a Surrogate Endpoint for Progression-free Survival in Newly Diagnosed Multiple Myeloma Studies: A Meta-analysis

BackgroundTherapeutic advances have greatly extended survival times in patients with multiple myeloma, necessitating increasingly lengthy trials when using survival outcomes as primary endpoints. A surrogate endpoint that can more rapidly predict survival could accelerate drug development. We conducted a meta-analysis to evaluate minimal residual disease (MRD) status as a valid progression-free survival (PFS) surrogate in patients with newly diagnosed multiple myeloma (NDMM). Materials and MethodsWe searched abstracts in PubMed, The American Society of Hematology, and the European Hematology Association for “myeloma,” “minimal residual disease,” and “clinical trial.” Because of the need to evaluate the treatment effect on MRD response, only randomized studies for subjects with NDMM were included. Details on the MRD-tested populations were required. The meta-analysis was performed by principles outlined at the 2013 United States Food and Drug Administration workshop on MRD in acute myeloid leukemia.42 For samples that were not measured for MRD and within the subset specified for MRD assessment, their MRD status was imputed from the samples that had known MRD status. Patients that were excluded from planned MRD assessment were considered MRD-positive. ResultsSix randomized studies, representing 3283 patients and 2208 MRD samples, met analysis inclusion criteria. MRD negativity rates ranged from 0.06 to 0.70. The treatment effect on the odds ratio for MRD-negative response strongly correlated with the hazard ratio for PFS with a coefficient of determination for the weighted regression line of 0.97. Our meta-analysis suggested that MRD status met both the Prentice criteria for PFS surrogacy. ConclusionsThese results support the claim that MRD status can be used as a surrogate for PFS in NDMM.

1409P - Known and unknown gene fusion detection capabilities of solid tumour laboratories conducting next generation sequencing in 6 countries

BackgroundDevelopments in next generation sequencing (NGS) have revolutionized oncogenic biomarker detection, removing the limitations of single-nucleotide polymorphisms (SNPs) to allow for massive parallel sequencing to detect multiple oncogenic gene fusions. We believe NGS testing platforms are not uniform within the current solid tumor lab landscape, with NGS analysis capabilities ranging from SNPs only to SNPs and known gene fusions, potentially missing crucial biomarkers. The objective of this study was to determine the current NGS capability of solid tumor labs to detect unknown fusions. MethodsThis study used real-world clinical pathology solid tumor labs data from the United States, United Kingdom, France, Spain, Italy, and Germany that conduct NGS. The Diaceutics proprietary global database of more than 2500 labs was analysed across these markets in Q4 2018 to understand NGS testing capabilities currently available within the routine clinical lab setting. ResultsThe results of the study are presented in the table. Of the 131 labs in the US performing NGS, only 50 were able to detect fusions. Of those 50 labs, 42 were restricted to using panels designed to detect specific known fusions. Only 8 labs in the US were able to detect unknown fusions. This pattern was reflected in labs surveyed in 5 other countries.Table: 1409PSolid tumor labs: NGS panel use and gene fusion detectionTable: 1409PCountryNumber of solid tumor labs performing NGSNumber of labs that can detect fusionsNumber of labs that can detect known fusionsNumber of labs that can detect known and unknown fusionsUS13150428France391174Germany21541Spain23440Italy30761UK24716 ConclusionsLabs are not well equipped to handle the pace of advancement in biomarker testing. While targeted treatments and companion diagnostics usher in a new age of precision medicine in cancer and extend survival times, many labs lack the technology needed to accurately identify emerging gene fusion biomarkers, especially unknown fusions. This results in both confusion among physicians regarding testing adequacy and missed opportunities for improved outcomes among patients. Legal entity responsible for the studyDiaceutics. FundingDiaceutics. DisclosureS. Finucane: Full / Part-time employment: Diaceutics. S. Haridas: Shareholder / Stockholder / Stock options, Full / Part-time employment: Diaceutics. L. Handley: Full / Part-time employment: Diaceutics. J. Clark: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Diaceutics. A. Jack: Shareholder / Stockholder / Stock options, Full / Part-time employment: Diaceutics. S. Munksted: Shareholder / Stockholder / Stock options, Full / Part-time employment: Diaceutics.

1219P - Maternal immunization against ALK as a weapon to fight neuroblastoma

BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in infancy. Some germline and somatic mutations associated with NB have been identified as activating mutations of the anaplastic lymphoma kinase (ALK) oncogene. Because of the nature of NB, which can occur in the early post-natal life or even during fetal life, we evaluated the efficacy of a DNA vaccine against ALK in a spontaneous preclinical model of NB, harboring ALKF1174L mutation in association with MYCN amplification (ALKF1174L/MYCN mice) by means of maternal immunization (MI). MethodsMYCN transgenic females were immunized against ALK by DNA vaccination by using a plasmid coding for the extracellular and trans-membrane domains of human ALK (ALK-ECTM) followed by electroporation, and then they were mated with an ALKF1174L-transgenic male. In ALKF1174L/MYCN offspring, the presence of abdominal, cervical and paraspinal tumors has been evaluated and quantified by Magnetic Resonance Imaging. The humoral immune response induced against ALK in the mothers and their offspring, as well as the presence of immune-complexes containing ALK, have been evaluated by ELISA. ALK expression in tumor tissue was assessed by Western blot. ResultsPre-birth immunization against ALK leads to an extended survival time and to a lower tumor growth kinetic in ALKF1174L/MYCN offspring born from ALK-ECTM-vaccinated mothers (ALK-ECTM offspring) as compared to controls born from control empty vector-vaccinated mothers. Maternally derived anti-ALK antibodies were successfully transferred from mothers to newborns. Moreover, anti-ALK IgM were found in the sera of five- and six-week old ALK-ECTM offspring, suggesting the induction of the pups’ own immune response against ALK. This effect could be due to the breast milk-mediated transfer of immune-complexes containing ALK, found in the milk of vaccinated mothers and in their offspring sera. Finally, MI against ALK induces a decrease in ALK expression in ALK-ECTM offspring’ tumor tissue. ConclusionsOverall, these results indicate that MI against ALK induces an active immunization against this oncoantigen in the offspring, impairing tumor development and enhancing survival time in a preclinical model of NB. Legal entity responsible for the studyDepartment of Molecular Biotechnology and Health Sciences. FundingAssociazione Italiana per la ricerca sul Cancro. DisclosureAll authors have declared no conflicts of interest.

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F

ObjectivesBotulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. MethodsIn present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model. ResultsThree compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (KD: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC50 of 30.47 ± 6.24, 14.91 ± 2.49 and 17.39 ± 2.74 μM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model. ConclusionNSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.

Metal-Organic Framework Encapsulating Hemoglobin as a High-Stable and Long-Circulating Oxygen Carriers to Treat Hemorrhagic Shock.

As an oxygen-transporting protein, free hemoglobin (Hb) often suffers from the disadvantage of undesirable stability and short blood circulation, which severely impairs the potential clinical applications as the blood substitute. In this work, Hb was facilely encapsulated into a kind of metal-organic frameworks (MOFs) (ZIF-8) inspired by the natural biomineralization process. The obtained ZIF-8 encapsulating Hb (ZIF-8@Hb) showed the small hydrodynamic size of 180.8 nm and neutral zeta potential of -2.1 mV by adjusting the ratio of Hb in ZIF-8 frameworks. Intriguingly, Hb encapsulated by ZIF-8 exhibited significantly enhanced stability in alkaline, oxidation, high temperature, or enzymatic environment compared with free Hb because of the excellent protective MOF coatings. More importantly, the negative charge of Hb neutralized the original positive charge of ZIF-8, which led to the better biocompatibility, lower protein adsorption, and macrophage uptake of ZIF-8@Hb than bare ZIF-8 nanoparticles. Furthermore, ZIF-8@Hb displayed extended blood circulation with the elimination half-life of 13.9 h as well as reduced nonspecific distribution in normal organs compared with free Hb or ZIF-8 nanoparticles. With the above advantages, ZIF-8@Hb showed significantly extended survival time of mice in a disease model of hemorrhagic shock compared with free Hb or bare ZIF-8 nanoparticles. Overall, this work offers a high-stable and long-circulating oxygen carrier platform, which may find wide applications as a blood substitute to treat various oxygen-relevant diseases.

Zespół odstawienia ruksolitynibu u chorej leczonej z powodu mielofibrozy

As an inhibitor of JAK1/JAK2 kinase, ruxolitinib is an effective drug for the treatment of patients with primary and secondary myelofibrosis. Around half such patients demonstrate recovery from the disease’s general symptoms and have their enlarged spleens reduced, together with having an extended survival time. Nevertheless, some patients suffer side effects which thus indicate that the drug is discontinued. In some cases a sudden discontinuation leads to ruxolitinib withdrawal syndrome presenting as a rapid relapse back to the disease symptoms before treatment, as well as rapid splenomegaly. Also rarely occurring, are severe haemodynamic disturbances requiring intensive care. We hereby present a case report of a patient in whom ruxolitinib was discontinued resulting in withdrawal syndrome appearance.

Antisense oligonucleotides extend survival of prion-infected mice.

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering 1 target mRNA in the brain, but their development for prion disease has been hindered by 3 unresolved issues from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here, we test ASOs delivered by bolus intracerebroventricular injection to intracerebrally prion-infected WT mice. Prophylactic treatments given every 2–3 months extended survival times 61%–98%, and a single injection at 120 days after infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a nontargeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent — or even single — bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.

Understanding extinction debts: spatio–temporal scales, mechanisms and a roadmap for future research

Extinction debt refers to delayed species extinctions expected as a consequence of ecosystem perturbation. Quantifying such extinctions and investigating long‐term consequences of perturbations has proven challenging, because perturbations are not isolated and occur across various spatial and temporal scales, from local habitat losses to global warming. Additionally, the relative importance of eco‐evolutionary processes varies across scales, because levels of ecological organization, i.e. individuals, (meta)populations and (meta)communities, respond hierarchically to perturbations. To summarize our current knowledge of the scales and mechanisms influencing extinction debts, we reviewed recent empirical, theoretical and methodological studies addressing either the spatio–temporal scales of extinction debts or the eco‐evolutionary mechanisms delaying extinctions. Extinction debts were detected across a range of ecosystems and taxonomic groups, with estimates ranging from 9 to 90% of current species richness. The duration over which debts have been sustained varies from 5 to 570 yr, and projections of the total period required to settle a debt can extend to 1000 yr. Reported causes of delayed extinctions are 1) life‐history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions. Other potential factors that may extend survival time such as microevolutionary dynamics, or delayed extinctions of interaction partners, have rarely been analyzed. Therefore, we propose a roadmap for future research with three key avenues: 1) the microevolutionary dynamics of extinction processes, 2) the disjunctive loss of interacting species and 3) the impact of multiple regimes of perturbation on the payment of debts. For their ability to integrate processes occurring at different levels of ecological organization, we highlight mechanistic simulation models as tools to address these knowledge gaps and to deepen our understanding of extinction dynamics.

Effects of Cecropin Transgenesis and Interspecific Hybridization on the Resistance to Ichthyophthirius multifiliis in Channel Catfish and Female Channel Catfish × Male Blue Catfish Hybrids

AbstractThe resistance of Channel Catfish Ictalurus punctatus, hybrid catfish (female Channel Catfish × male Blue Catfish I. furcatus [CB hybrids]), and Channel Catfish and hybrid catfish expressing the introduced cecropin B gene to Ichthyophthirius multifiliis infestation was investigated in two experiments. In experiment I, four fingerling groups were challenged, including cecropin‐transgenic Channel Catfish, cecropin‐transgenic CB hybrids, non‐transgenic Channel Catfish, and non‐transgenic CB hybrids. Non‐transgenic Channel Catfish survived for a shorter time than the other three groups. Survival rate was significantly different between non‐transgenic Channel Catfish and the other groups, which had similar survival rates. In experiment II, non‐transgenic CB hybrids had a less severe infestation than non‐transgenic Channel Catfish. Mortality rates were 62.4% and 40.2% for non‐transgenic Channel Catfish and CB hybrids, respectively. The mean survival time for non‐transgenic hybrids was significantly longer (&gt;5 d) than that of non‐transgenic Channel Catfish. The results suggest that genetic enhancement of Ichthyophthirius resistance can be accomplished in Channel Catfish by either cecropin transgenesis or interspecific hybridization. In addition to survival rate, improving survival time is important because the extension of survival time provides greater opportunity to apply treatments to stop the protozoan infestation.

The encouraging role of long noncoding RNA small nuclear RNA host gene 16 in epithelial-mesenchymal transition of bladder cancer via directly acting on miR-17-5p/metalloproteinases 3 axis.

The present investigation was intended to elucidate whether long noncoding RNA small nuclear RNA host gene 16 (SNHG16) could regulate the epithelial-mesenchymal transition process of bladder cancer cells by directing expressions of miR-17-5p and metalloproteinases 3 (TIMP3). To elucidate the point, we collected 275 pairs of bladder cancer tissues and corresponding adjacent normal tissues, as well as four bladder cancer cell lines and the normal human bladder epithelial cell line. Moreover, pcDNA3.1-SNHG16, si-SNHG16, miR-17-5p mimic, miR-17-5p inhibitor, pcDNA3.1-TIMP3, and si-TIMP3 were prepared for transfection, and CCK-8 assay, colony formation assay, flow cytometry, wound healing assay, and transwell assay were carried out. Finally, the dual luciferase reporter gene assay was performed to figure out whether targeted regulations were present among SNHG16, miR-17-5p, and TIMP3. The laboratory findings demonstrated that the bladder cancer patients carrying under-expressed SNHG16 or miR-17-5p were associated with extended survival time when compared with those possessing overexpressed SNHG16 and miR-17-5p (P < 0.05). Furthermore, overexpression of SNHG16 and miR-17-5p both enhanced the viability, proliferation, migration, and invasion (P < 0.05), and simultaneously suppressed their apoptosis (P < 0.05). Transfections of pcDNA3.1-SNHG16 and si-SNHG16, respectively, resulted in overexpression and under-expression of miR-17-5p, and the dual luciferase reporter gene assay demonstrated a targeted relationship between SNHG16 and miR-17-5p (P < 0.05). Besides, the expression of TIMP3 was subjected to targeted regulation of miR-17-5p (P < 0.05), and its overexpression could reverse the effects of miR-17-5p on proliferation and metastasis (P < 0.05). Conclusively, purposeful modification of SNHG16/miR-17-5p/TIMP3 signaling might be conducive to postpone the aggravation of bladder cancer.