C57B1 Mice Research Articles

Structure-Function Correlation in Cobalt-Induced Brain Toxicity.

Cobalt toxicity is difficult to detect and therefore often underdiagnosed. The aim of this study was to explore the pathophysiology of cobalt-induced oxidative stress in the brain and its impact on structure and function. Thirty-five wild-type C57B16 mice received intraperitoneal cobalt chloride injections: a single high dose with evaluations at 24, 48, and 72 h (n = 5, each) or daily low doses for 28 (n = 5) or 56 days (n = 15). A part of the 56-day group also received minocycline (n = 5), while 10 mice served as controls. Behavioral changes were evaluated, and cobalt levels in tissues were measured with particle-induced X-ray emission. Brain sections underwent magnetic resonance imaging (MRI), electron microscopy, and histological, immunohistochemical, and molecular analyses. High-dose cobalt caused transient illness, whereas chronic daily low-dose administration led to long-term elevations in cobalt levels accompanied by brain inflammation. Significant neurodegeneration was evidenced by demyelination, increased blood-brain barrier permeability, and mitochondrial dysfunction. Treated mice exhibited extended latency periods in the Morris water maze test and heightened anxiety in the open field test. Minocycline partially mitigated brain injury. The observed signs of neurodegeneration were dose- and time-dependent. The neurotoxicity after acute exposure was reversible, but the neurological and functional changes following chronic cobalt administration were not.

Regeneration of the cerebral cortex by direct chemical reprogramming of macrophages into neuronal cells in acute ischemic stroke.

Theoretically, direct chemical reprogramming of somatic cells into neurons in the infarct area represents a promising regenerative therapy for ischemic stroke. Previous studies have reported that human fibroblasts and astrocytes transdifferentiate into neuronal cells in the presence of small molecules without introducing ectopic transgenes. However, the optimal combination of small molecules for the transdifferentiation of macrophages into neurons has not yet been determined. The authors hypothesized that a combination of small molecules could induce the transdifferentiation of monocyte-derived macrophages into neurons and that the administration of this combination may be a regenerative therapy for ischemic stroke because monocytes and macrophages are directly involved in the ischemic area. Transcriptomes and morphologies of the cells were compared before and after stimulation using RNA sequencing and immunofluorescence staining. Microscopic analyses were also performed to identify cell markers and evaluate functional recovery by blinded examination following the administration of small molecules after ischemic stroke in CB-17 mice. In this study, an essential combination of six small molecules [CHIR99021, Dorsomorphin, Forskolin, isoxazole-9 (ISX-9), Y27632, and DB2313] that transdifferentiated monocyte-derived macrophages into neurons in vitro was identified. Moreover, administration of six small molecules after cerebral ischemia in model animals generated a new neuronal layer in the infarct cortex by converting macrophages into neuronal cells, ultimately improving neurological function. These results suggest that altering the transdifferentiation of monocyte-derived macrophages by the small molecules to adjust their adaptive response will facilitate the development of regenerative therapies for ischemic stroke.

Therapeutic Performance Evaluation of 213Bi-Labelled Aminopeptidase N (APN/CD13)-Affine NGR-Motif ([213Bi]Bi-DOTAGA-cKNGRE) in Experimental Tumour Model: A Treasured Tailor for Oncology

Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 μL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.

Induction of angiogenesis and neural progenitor cells by basic fibroblast growth factor-releasing polyglycolic acid sheet following focal cerebral infarction in mice.

Biodegradable sheets loaded with basic fibroblast growth factor (bFGF) are prepared as novel bFGF-releasing systems from polyglycolic acid nonwoven fabric by oxygen plasma treatment followed by bFGF adsorption. In the present study, we investigated the therapeutic effects of this system on a focal cerebral infarction model (CB-17 mouse). A preliminary in vitro study showed that this system released bFGF in an acellular culture medium, thereby keeping the bFGF concentration in the medium at ≥5ng/ml for a prolonged period of 7 days. The released bFGF from this system retained its biological activity to enhance endothelial tube formation in vitro. In a mouse model of subacute focal cerebral infarction, this system increased the expression of endogenous vascular endothelial growth factor in the peri-infarct cortex and subventricular zone, promoted angiogenesis in the striatum, and increased neural progenitor cells in the peri-infarct cortex. Thus, this bFGF-releasing system has the potential to be a novel therapeutic approach for cerebral infarction.

Calcitonin gene-related peptide exerts inhibitory effects on autophagy in the heart of mice

Calcitonin Gene-Related Peptide (CGRP) is a potent vasodilator peptide widely distributed in the central nervous system and various peripheral tissues, including cardiac muscle. However, its role in heart protein metabolism remains unknown. We examined the acute effects of CGRP on autophagy and the related signaling pathways in the heart mice and cultured neonatal cardiomyocytes. CGRP (100 μg kg−1; s.c.) or 0.9 % saline was injected in awake male C57B16 mice, and the metabolic profile was determined up to 60 min. In fed mice, CGRP drastically increased glycemia and reduced insulinemia, an effect that was accompanied by reduced cardiac phosphorylation levels of Akt at Ser473 without affecting FoxO. Despite these catabolic effects, CGRP acutely inhibited autophagy as estimated by the decrease in LC3II:LC3I and autophagic flux. In addition, the fasting-induced autophagic flux in mice hearts was entirely abrogated by one single injection of CGRP. In parallel, CGRP stimulated PKA/CREB and mTORC1 signaling and increased the phosphorylation of Unc51-like kinase-1 (ULK1), an essential protein in autophagy initiation. Similar effects were observed in cardiomyocytes, in which CGRP also inhibited autophagic flux and stimulated Akt and FoxO phosphorylation. These findings suggest that CGRP in vivo acutely suppresses autophagy in the heart of fed and fasted mice, most likely through the activation of PKA/mTORC1 signaling but independent of Akt.

Analgesic effect of methanolic extract obtained from Schinus terebinthifolius leaves

Previous studies from our group showed the analgesic effects of essential oil and extracts of Schinus terebinthifolius. The aims of this research were to evaluate the anti-nociceptive, anti-arthritic properties of methanolic extract obtained from S. terebinthifolius (MEST) leaves in animal models of experimental pain and arthritis. The MEST (30 and 100 mg/kg) reduced both phases of formalin induced licking behavior in Swiss mice. The MEST (100 mg/kg) reduced mechanical hyperalgesia induced by carrageenan in swiss mice. The MEST (100 mg/kg) oral administration reduced significantly the mechanical hyperalgesia (day 2, 5, 10, and 20), knee oedema (day 2), cold (day 15) and heat sensitivity (day 20), but not depressive-like behavior (Forced Swim Test), after Complete Freund's Adjuvant (CFA) in C57B16 mice. The MEST prevented the anti-nociceptive effects in formalin nociception and did not interfere with locomotor activity in Swiss mice (open field test). The MEST was effective for the inhibition of pain and arthritic parameters without altering locomotor activity or depressive-like behavior. The present results may open new possibilities for the development of new analgesic agents from S. terebinthifolius.

Abstract WP152: Novel Therapy With Pdgf-b Nanoparticles for Cerebral Infarction

Background and Purpose: Treatment for cerebral infarction beyond the indication of reperfusion therapy has not yet been established, and novel approaches are needed. It has been reported that platelet-derived growth factor (PDGF)-B played a neuroprotective role by enhancing cell survival and tissue repair for a long period after cerebral infarction in experimental animal models. However, PDGF-B is difficult to administer at effective concentrations in infarct area. In general, nanoparticles are very small and stable, making them prone to accumulation without being metabolized in infarct area. Therefore, we converted PDGF-B into nanoparticles and examined its therapeutic effect for cerebral infarction. Methods: PDGF-B nanoparticles (PDGF-B NP) and wild-type nanoparticles (wNP) were injected one day after transient middle cerebral artery occlusion (tMCAO) using the CB-17 mouse model. We analyzed temporal histological changes and neurological function recovery. Fucntional recovery was assess using Cylinder test at 3 and 7 days after the tMCAO. As for the mechanism of neuroprotective effect, phosphorylation of Akt, neurotrophin-3 (NT-3), and expression of angiogenesis were also examined in the infarct area and compared them with wNP control at 7 days after tMCAO. Result: We found that PDGF-B NP was distributed specifically in the infarct area. As compared with wNP group, PDGF-B NP group significantly suppressed cerebral infarct volume and improved neurological function at 3 and 7 days after cerebral infarction compared with wNP group. Akt was strongly phosphorylated in the infarction area with PDGF-B NP administration compared with wNP. Moreover, PDGF-B NP significantly induced angiogenesis, NT-3 expression, and reduced cell apoptosis after cerebral infarction compared with wNP. Conclusion: PDGF-B NP activated PDGF-B-Akt signaling in infarct area and played various important roles leading to neuroprotection after cerebral infarction. Our results suggested that treatment with PDGF-B NP may be useful for cerebral infarction beyond reperfusion therapy.

Abstract 1778: 5’-nucleotidases are involved in the biology of human lung cancer cell lines

Abstract Cytosolic-5’-nucleotidase II (cN-II) and ecto-5′-nucleotidase (CD73) are enzymes involved in the nucleotide metabolism by dephosphorylating intracellular and extracellular purine nucleotide monophosphates, respectively. Both enzymes have been shown to be involved in cancer by modifying anticancer drug activity, cancer cell biology and immune modulation. To increase the knowledge of cN-II and CD73 roles in cancer cell biology, we developed lung cancer cell models (NCI-H292) with a complete knockout of either or both enzymes using the CRISPR-Cas9 technique. These cell models are used to study cell proliferation and migration, cancer drug sensitivity, cellular behaviour and apoptosis using CFSE staining, IncuCyte relative confluence and wound healing assay, MTT assay, xCELLigence RTCA and caspase-3/7 apoptotic assay. SCID CB17 mice are also used to study the in vivo tumor growth using these cell models. Our results show that there is no significant difference in proliferation between different cell models exposed or not to different concentrations of either adenosine or AMP under normoxic and hypoxic conditions. On the other hand, using xCELLigence RTCA technique, we have observed that CD73-deficient cells have higher cell index as compared to CD73 expressing cells under basal conditions during the first 72 hours of culture. However, adenosine globally decreases the cell index of all NCI-H292 cells, unlike AMP, which influences the cell index of cN-II-deficient cells but not CD73-deficient cells. Under hypoxic condition, cN-II-deficient cells showed higher cell index than the other phenotypes in untreated conditions, whereas cells lacking CD73 showed higher sensitivity to adenosine or AMP. As compared to other phenotypes, CD73 deficient cells are also more sensitive towards vincristine but less sensitive towards mitomycin and fludarabine. These cells are also more prone to adapt to hypoxic conditions and exhibit slower migration rate than their corresponding control cells. However, the effect of adenosine and AMP on migration rate is higher on cN-II-deficient cells. Mice experiments showed no difference in in vivo tumor growth using these cell models. Overall, we show that both CD73 and cN-II are important players in the cell biology of lung cancer cells and in their response to purines. Upcoming experiments should help us understand the molecular mechanisms underlying these observed differences. Citation Format: Muhammad Zawwad Raza, Octavia Cadassou, Emeline Cros-Perrial, Alain Puisieux, Charles Dumontet, Lars Petter Jordheim. 5’-nucleotidases are involved in the biology of human lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1778.

Early initiation of a factor Xa inhibitor can attenuate tissue repair and neurorestoration after middle cerebral artery occlusion

The timing of anti-coagulation therapy initiation after acute cardioembolic stroke remains controversial. We investigated the effects of post-stroke administration of a factor Xa inhibitor in mice, focusing on tissue repair and functional restoration outcomes. We initiated administration of rivaroxaban, a Xa inhibitor, immediately after permanent distal middle cerebral artery occlusion (pMCAO) in CB-17 mice harboring few leptomeningeal anastomoses at baseline. Rivaroxaban initiated immediately after pMCAO hindered the recovery of blood flow in ischemic areas by inhibiting leptomeningeal anastomosis development, and led to impaired restoration of neurologic functions with less extensive peri-infarct astrogliosis. Within infarct areas, angiogenesis and fibrotic responses were attenuated in rivaroxaban-fed mice. Furthermore, inflammatory responses, including the accumulation of neutrophils and monocytes/macrophages, local secretion of pro-inflammatory cytokines, and breakdown of the blood–brain barrier, were enhanced in infarct areas in mice treated immediately with rivaroxaban following pMCAO. The detrimental effects were not found when rivaroxaban was initiated after transient MCAO or on day 7 after pMCAO. Collectively, early post-stroke initiation of a factor Xa inhibitor may suppress leptomeningeal anastomosis development and blood flow recovery in ischemic areas, thereby resulting in attenuated tissue repair and functional restoration unless occluded large arteries are successfully recanalized.

Renal Sensory Nerves Increase Blood Pressure and Sympathetic Nerve Activity in 2‐Kidney 1‐Clip Hypertensive Mice

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to determine whether renal sensory nerves contribute to the 2‐Kidney‐1‐Clip (2K1C) model of hypertension. 2K1C hypertension was produced in male C57B16 mice (12–14 weeks, Jackson Laboratories) by placement of a 0.5mm length of PTFE tubing (ID: 0.008” x OD: 0.014”) around the left renal artery. 2K1C mice (n=10) displayed an elevated ABP measured via telemetry (Day 0: 96 ± 1mmHg vs Day 14: 120 ± 3mmHg; P<0.01). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 14 vs Day 0 (Day 0: −35 ± 2mmHg vs Day 14: −57 ± 5mmHg; P<0.01). 2K1C‐clipped kidneys vs 2K1C‐unclipped kidneys had lower mass (105 ± 14mg vs 173 ± 8mg, respectively; P<0.05) and higher mRNA levels of several pro‐inflammatory cytokines (IL1B, IL2, IL10, TNFa; P<0.05). Renal afferent discharge was significantly higher in 2K1C‐clipped kidneys vs 2K1C‐unclipped vs sham mice (2K1C‐clipped: 3.4 ± 0.9 Hz vs 2K1C‐unclipped: 0.7 ± 0.3Hz vs control: 0.3 ± 0.1 Hz, n=6/group; P<0.05). Furthermore, electrical stimulation of renal afferent nerves in control mice (n=6) produced a frequency‐dependent increase in ABP (5Hz: 4 ± 1mmHg, 10Hz: 6 ± 1mmHg, 20Hz: 11 ± 2mmHg; P<0.05). These responses were eliminated after ganglionic blockade with 5mg/kg chlorisondamine (5Hz: 1 ± 1mmHg, 10Hz: 1 ± 1mmHg, 20Hz: 1 ± 1mmHg, n=4/group; P<0.05). Stimulus‐triggered averaging of SNA during stimulation of renal afferent nerves (1 Hz, 200uA) revealed significant (P<0.05, n=4/group) increases in splanchnic (179 ± 11%), renal (195 ± 15%), and lumbar (167 ± 9%) SNA. Interestingly, the latency to the peak SNA (142 ± 10ms) suggests supraspinal pathways mediate the sympathoexcitatory response. Finally, both total renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33mM capsaicin) at time of clipping resulted in lower ABP than 2K1C mice at Day 14 (2K1C: 120 ± 3mmHg, phenol: 106 ± 3mmHg, capsaicin: 108 ± 3mmHg, n=6–10/group; P<0.05). These findings suggest 2K1C hypertension depends on renal sensory nerves and elevated SNA via supraspinal pathways.Support or Funding InformationNIH grant HL113270, AHA Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell-intrinsic PD-L1 and PD-1 signal effects in bladder cancer

Abstract PD-1/PD-L1 provides a mechanism of immune escape, the blockage of which has reinvigorated interest in the treatment of urothelial cancer. We recently reported on cell-intrinsic PD-L1 effects in melanoma and ovarian cancer, and considered that effects in bladder cancer cells could differ based on distinct mutational landscapes. Using CRISPER/Cas9 methodology, we knockout PD-L1 in the murine bladder cancer cell line MB49 and the human bladder caner cell line RT. We show here that cell-intrinsic MB49 and RT4 cells express PD-L1 and PD-1 that each mediate cell-intrinsic signals. PD-L1 knockout has little effect on cells proliferation in vitro, but significant difference could be found in tumor size when we used MB49 Ctrl or MB49 PD-L1KO cells to challenge C57B16 mice. αPD-L1 and αPD-1 antibodies reduced bladder cancer cell proliferation in vitro demonstrating direct signaling effects. Bladder cancer cell-intrinsic PD-L1 promoted mTORC1 and tumor initiating cell generation similar to melanoma and ovarian cancer cells. By contrast to melanoma and ovarian cancer cells, bladder cancer cell PD-L1 promoted autophagy and had little effect on in vivo immune-independent growth. Base one the functional role, PD-L1 knockout increased proliferation inhibition induced by Rapamycin in both mouse and human bladder cancer cells. Furthermore, we found intrinsic PD-L1 suppress cytokine production, including CXCL10. Corresponding with increased CXCL10 secretion, more CXCR3+ NK cells could be attracted by PD-L1KO cancer cells. Overall, our findings further illustrated the intrinsic role of PD-L1 in bladder cancer development and found PD-1 expression on human bladder cancer cells for the first time.

Effects of nano-encapsulated curcumin-chrysin on telomerase, MMPs and TIMPs gene expression in mouse B16F10 melanoma tumour model

Due to the high rate of drug resistance among malignant melanoma cases, it seems necessary to introduce an efficient pharmaceutical approach to melanoma treatment. For this purpose, Curcumin (Cur) and Chrysin (Chr), two natural anti-cancers, were co-encapsulated in PLGA-PEG nanoparticles (NPs), characterized by DLS, FTIR and FE-SEM and investigated for their effects on MMPs, TIMPs and TERT genes expression in C57B16 mice bearing B16F10 melanoma tumours. The results showed that the expression of MMP-9, MMP-2 and TERT genes were significantly decreased in all treated groups compared to the control. This reduction had the highest amount in CurChr NPs group and then CurChr group for each three genes. Likewise, the expression of TIMP-1 and TIMP-2 genes was significantly increased in all treated groups, compared to the control. Combination groups showed the highest rise in expression of these two genes and the observed increase was greater in nano groups. Moreover, the highest melanoma tumour growth inhibition was detected for CurChr NPs, followed by CurChr = Cur NPs > Cur > Chr NP > Chr. Overall, it is speculated that the nano-combination of Cur and Chr into polymeric NPs with a one-step fabricated co-delivery system may be a promising and convenient approach to improve their efficiency in melanoma cancer therapy.

Differences of Matrix Metalloproteinase 2 Expression between Left and Right Ventricles in Response to Nandrolone Decanoate and/or Swimming Training in Mice.

Background:Matrix metalloproteinase (MMP)-2 plays an important role in the remodeling of left ventricles (LVs) and right ventricles (RVs). We investigated the differences of MMP-2 expression between LV and RV in response to nandrolone decanoate (ND), swimming training (ST), and combined ND and ST (NS) in mice, based on their structural, functional, and biochemical characteristics.Methods:Totally 28 male C57B1 mice (6 weeks old; 20–23 g) were divided into four groups, including the control (n = 7), ND (n = 6), ST (n = 8), and NS (n = 7) groups. After respective treatments for 8 weeks, echocardiographic examination was used to assess the cardiac structure and function. Van Gieson stain was used to examine the fibrosis of LV and RV in response to different treatments, and Western blotting analysis was performed to explore different MMP-2 expressions between LV and RV in response to ND and/or ST. Analysis of variance was used for comparing the four groups.Results:At 8 weeks, right ventricular dimension/body weight in the ND group was larger than the other three groups (F = 7.12, P < 0.05) according to the echocardiographic examination. Fibrosis induced by ND administration was increased more in RV (2.59%) than that in LV (2.21%). MMP-2 expression of the ND group in RV was significantly greater than the control and NS groups in RV and the corresponding ND group in LV.Conclusion:The experimental data support the hypothesis that ND administration induces greater MMP-2 expression increase in RV compared to LV, leading to consequent RV dilation.

Evaluation of oxidative stress, antioxidant enzyme activities, and genetic damage in sprayers exposed to pesticides

The potential immunotoxic effect of the carbamate pesticide aminocarb on murine bone marrow cell subpopulations was evaluated by flow cytometry, C57B16 mice were exposed by gavage to sublethal doses of the pesticide and lymphocyte precursors from bone marrow population were stained with PNA lectin and a panel of monoclonal antibodies against cell surface antigens. In regard to the microenvironment-dependent maturation of B lymphocytes, the pesticide effect on the lymphoproliferative potential of bone marrow was assessed by marrow transplantation from aminocarb-exposed donor mice to normal, syngeneic, X-irradiated recipient mice. The sublethal exposure of 0.08–5.0 mg/kg body wt aminocarb to donor animals did not affect regenerating bone marrow in the recipient mice. No marked effect on bone marrow cell number was noted in pesticide-exposed animals. However, a marked shift in surface IgM density on marrow B cells was noted at 0.08 and 0.31 mg/kg body wt aminocarb. This was correlated with decreased cell frequency in G0G1 phase and increased frequency of cells in the S phase of the cell cycle. Thus, altered maturation of B lymphocytes, expressed as a shift in the density of surface IgM on mature B cells and not the lymphocyte count, was related to the direct effect of aminocarb and/or to the pesticide-related changes in bone marrow microenvironment. Overall, exposure to the carbamate pesticide aminocarb activated the cell maturation process in bone marrow.

Ferritin surplus in mouse spleen 14 months after intravenous injection of iron oxide nanoparticles at clinical dose

In this study, we followed the biodegradation of ultra-small superparamagnetic iron oxide nanoparticles injected intravenously at clinical doses in mice. An advanced fitting procedure for magnetic susceptibility curves and low-temperature hysteresis loops was used to fully characterize the magnetic size distribution as well as the magnetic anisotropy energy of the injected P904 nanoparticles (Guerbet Laboratory). Additional magnetometry measurements and transmission electronic microscopy observations were systematically performed to examine dehydrated samples from the spleen and liver of healthy C57B16 mice after nanoparticle injection, with sacrifice of the mice for up to 14 months. At 3 months after injection, the magnetic properties of the spleen and liver were dramatically different. While the liver showed no magnetic signals other than those also present in the reference species, the spleen showed an increased magnetic signal attributed to ferritin. This surplus of ferritin remained constant up to 14 months after injection.

Xenon Confers Neuroprotection in Organotypic Hippocampal Slice Cultures

Background: The present study sought to establish the use of a novel model of cerebral ischaemia in which to investigate the neuroprotective effects of xenon based on organotypic hippocampal slice cultures.Methods: Organotypic hippocampal slices, derived from 7-10 days post-natal C57B16 mouse pups, were placed and cultured for 10-14 days on membranous inserts. Neuronal injury of cultures was provoked with media deprived with oxygen and glucose (OGD) for 2 hours. Cultures were exposed to 25% xenon or 50% xenon and 20% oxygen and 5% carbon dioxide (CO2) balanced with nitrogen (N2) 24 hours before OGD insult (preconditioning) for 2 hours or 75% xenon and 5% CO2 balanced with oxygen 4 hours after OGD for 2 hours. Cell death was quantified by assessment of propidium iodide (PI) uptake before and after OGD up to 72 hours in the CA1 and the dentate gyrus (DG) of the hippocampus.Results: Xenon preconditioning at concentrations of 25% and 50% resulted in a significant reduction in PI uptake in the CA1 region of the hippocampus compared with the control group (P<0.01). The effect was less pronounced in the DG region with xenon only at a concentration of 25% (P<0.05). Xenon 75% treatment 4 hours post-insult significantly attenuated cell death in both the DG and CA1 areas of the hippocampus.Conclusions: This study corroborated previous data to confirm that xenon is a potent neuroprotectant, and advocated its potential for future use in specific clinical settings. Citation: Fei Lan, Sheena Sodha, Tian-Long Wang, Da-Qing Ma. Xenon confers neuroprotection in organotypic hippocampal slice cultures. J Anesth Perioper Med 2016; 3: 63-9. doi: 10.24015/JAPM.2016.0009This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Comparative immunogenicity and structural analysis of epitopes of different bacterial L-asparaginases.

BackgroundE.coli type II L-asparaginase is widely used for treatment of acute lymphoblastic leukemia. However, serious side effects such as allergic or hypersensitivity reactions are common for L-asparaginase treatment. Methods for minimizing immune response on L-asparaginase treatment in human include bioengeneering of less immunogenic version of the enzyme or utilizing the homologous enzymes of different origin. To rationalize these approaches we compared immunogenicity of L-asparaginases from five bacterial organisms and performed sequence-structure analysis of the presumable epitope regions.MethodsIgG and IgM immune response in C57B16 mice after immunization with Wollinella succinogenes type II (WsA), Yersinia pseudotuberculosis type II (YpA), Erwinia carotovora type II (EwA), and Rhodospirillum rubrum type I (RrA) and Escherichia coli type II (EcA) L-asparaginases was evaluated using standard ELISA method. The comparative bioinformatics analysis of structure and sequence of the bacterial L-asparaginases presumable epitope regions was performed.ResultsWe showed different immunogenic properties of five studied L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin as a second-line treatment. Studied L-asparaginases might be placed in the following order based on the immunogenicity level: YpA > RrA, WsA ≥ EwA > EcA. Most significant cross-immunogenicity was shown between EcA and YpA. We propose that a long N-terminus of YpA enzyme enriched with charged aminoacids and tryptophan could be a reason of higher immunogenicity of YpA in comparison with other considered enzymes. Although the recognized structural and sequence differences in putative epitope regions among five considered L-asparaginases does not fully explain experimental observation of the immunogenicity of the enzymes, the performed analysis set the foundation for further research in this direction.ConclusionsThe performed studies showed different immunogenic properties of L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin. The preferable enzymes for the second line treatment are WsA, RrA, or EwA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2125-4) contains supplementary material, which is available to authorized users.

A Novel Pan-PIM Kinase Inhibitor, SEL24-B489, Induces Apoptosis and Inhibits Proliferation of Diffuse Large B-Cell Lymphoma Cells through Inhibition of Protein Translation and Attenuation of Myc and NFkB Activity

Introduction. In 1/3of diffuse large B-cell lymphoma (DLBCL) patients current treatment options are ineffective, underscoring the need for more effective targeted therapies. Simultaneous genetic ablation of PIM kinases (PIM-1/2/3) or their pharmacological inhibition induce apoptosis in DLBCL cell lines, providing proof of concept that these proteins are promising targets in this disease. To better understand mechanisms of toxicity of PIM inhibition in DLBCL, we investigated biochemical and biological consequences of PIM inhibition using novel pan-PIM inhibitor (SEL24-B489) in a panel of DLBCL cell lines in in vivo xenograft model.Methods. Protein expression and phosphorylation status of PIM substrates was assessed by immunobloting. Proliferation and apoptosis were assessed by MTS assay and PI/AnnexinV staining. Gene expression profiling was performed on Illumina HT-12 v4 Chip. NFkB target genes expression was assessed by Real-Time Quantitative PCR (qPCR). For xenograft studies, 9-11 weeks old SCID/beige CB17 mice were subcutaneously injected with 5x106 U2932 cells suspended in 0.1 ml mixture of PBS with Matrigel. When tumor volumes reached ~100 mm3, mice were randomized into uniform groups and subjected to the compound administration (50 mg/kg daily, BID, per os). Tumor volume was monitored every second day.Results. The newly developed pan-PIM inhibitor (SEL24-B489) was toxic to DLBCL cell lines in low-micromolar or sub-micromolar concentrations (IC50: 0,29 to 1,17µM). To determine mechanisms underlying toxicity of PIM inhibition in DLBCL, we first assessed the activity of 4EBP1 and ribosomal protein S6, proteins involved in protein translation. PIM inhibitor rapidly and uniformly decreased 4EBP1 and S6 phosphorylation in all tested DLBCL cell lines (DHL4, DHL6, Ly1, Ly7, Karpas 422, Pfeiffer, Toledo, U2932, HBL-1). Since PIM kinases have been shown to increase stability and/or activity of NFκB-p65 (RelA) and Myc, we assessed the abundance/activity of these transcription factors upon PIM inhibitor treatment. PIM inhibition led to downregulation of Myc protein in GCB DLBCL cell lines. Since Myc and PIMs cooperate in augmenting transcription of active genes, we assessed global RNA levels upon PIM inhibitor treatment. The RNA abundance in SEL24-B489-treated cells decreased by 22-37%. The underlying mechanism of the decrease in cellular mRNA content involved global inhibition of PIM-dependent histone H3 serine 10 (H3S10) phosphorylation and decreased phoshorylation of RNA polymerase II (serine 2).To test whether PIM inhibition also attenuates specifically NFκB-dependent transcription in DLBCL cell lines, we assessed gene expression profiles of vehicle- or SEL24-B489-treated HBL-1, U2932 and DHL4 cells. Following PIM inhibition, we observed significant downregulation of multiple validated NFκB target genes in HBL-1 and U2932 cells, but not in DHL4 cells. We further confirmed decreased transcript abundance of 4 NFκB target genes (NFKBIA, CD40, TNFAIP 3 and MIR 155) by qPCR. Since NFκB target gene expression in ABC-DLBCL is essential for their survival and can be specifically blocked with the BTK inhibitor ibrutinib, we hypothesized that simultaneous targeting of PIMs and BTK would synergistically inhibit NFkB activity and cell survival. To test this hypothesis, we incubated the ABC-DLBCL cell lines with the ibrutinib, SEL24-B489, or their combination. We identified synergistic growth inhibitory effects of the drug combination in ABC-DLBCL cell lines, with combination index (CI) of 0.51 and 0.2 for HBL-1 and U2932 cells, respectively. Finally, we investigated the efficacy of SEL24-B489 in vivo in the murine xenograft model using U2932 cells. In contrast to progressive tumor growth in animals exposed to vehicle alone, we observed marked inhibition of tumor growth (>90%) in SEL24-B489-treated mice.Conclusions. In conclusion, a novel pan-PIM inhibitor SEL24-B489 induces apoptosis of DLBCL cell lines in low/sub-micromolar concentrations and exhibits activity in a xenograft model. The mechanisms of SEL24-B489 toxicity include blocking of protein translation, induction of Myc degradation, decrease of RNA transcription and attenuation of NFκB activity. Moreover, we show marked synergy between the PIM inhibitor and ibrutinib. Hence, these results provide new insights into mechanism of action of PIM inhibitors and rationale for targeting PIM activity in DLBCLs. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Golas:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.

Abstract 5014: Neutrophils protect B-cell lymphomas against chemotherapy via cell-cell interactions mediated by CD44 and ICAM1 receptors

Abstract Background: Neutrophils are innate immune cells that play an established role in the killing of microorganisms. While much less studied than adaptive immune cells, neutrophils are detected in different solid tumors. Current evidence suggests that neutrophils show high functional plasticity and can display either protumor or antitumor activity. Given the abundance of significant portion of tumor-associated neutrophils in many cancer models and due to lack of information about the role of neutrophils in lymphoma, we investigated the impact of neutrophils on the sensitivity of B cell lymphomas to chemotherapy. Methods: Freshly isolated human neutrophils were cocultured with different types of human B cell non Hodgkin lymphomas (NHL) in the presence of various chemotherapies (vincristine, doxorubicin, cisplatin, bortezomib and mafosfamide). Chemotherapeutic effect on cell proliferation and cytotoxicity was assessed by flow cytometry using CFSE and DAPI assays, respectively. Expression profiling of cells exposed to neutrophils was performed by Illumina cDNA array. Blocking antibodies were added to the coculture system. In vivo, RL cells were co-injected with neutrophils subcutaneously into SCID CB17 mice and treated with vincristine. Ex vivo, peripheral blood was obtained from chronic lymphocytic leukemia (CLL) patients. CLL B cells were cocultured with autologous neutrophils then exposed to vincristine, ibrutinib or idelalisib. CLL cells were analyzed for their content in Bcl2, BclXL and Mcl1. Results: We found that freshly isolated human neutrophils protect NHL cells against the chemotherapeutic cytotoxicity. Neutrophils protective role was not due to sequestration of chemotherapy. This effect was not due to soluble factors produced by neutrophils and required direct cell-cell interactions between neutrophils and NHL cells. Expression profiling of RL cells in the presence of neutrophils showed enhanced expression of CD44. Blocking antibodies against CD44 and ICAM1 receptors inhibit neutrophil-mediated protection of RL cells against chemotherapy in vitro. In vivo, freshly isolated human neutrophils co-injected with tumor cells reduced sensitivity of RL cells to vincristine resulting in enhanced tumor growth. Similarly autologous neutrophils protected fresh CLL cells from the cytotoxic effect of various agents. Exposure of CLL cells to neutrophils increased the content of Bcl2 and Mcl1 with no effect on BclXL. Conclusion: Neutrophils protect B cell lymphomas against chemotherapy in vitro and in vivo. This protective role is due to cell-cell interactions mediated by CD44 and ICAM1 receptors. Our results also suggest that neutrophils enhance CLL survival by inducing the expression of anti apoptotic proteins Bcl2 and Mcl1. Our findings underline the importance of neutrophils in tumor growth or chemoresistance and may contribute to therapeutic strategies to inhibit their protective effect. Citation Format: Taghreed Hirz, Justine Esmenjaud, Anne Evesque, Doriane Mathe-Poloni, Charles Dumontet. Neutrophils protect B-cell lymphomas against chemotherapy via cell-cell interactions mediated by CD44 and ICAM1 receptors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5014. doi:10.1158/1538-7445.AM2015-5014

Abstract W P98: Monocyte Chemotactic Protein-3 Elevated in Stoke and Aging

Background: It is now recognized that stroke is a systemic stressor that triggers profound changes throughout the body, leading to alterations in the immune system and response. In the CNS, cell death from ischemic stroke activates glial cells, leading to trafficking of leukocytes into the brain and subsequent inflammation. Chemokines play an active role in modulating this recruitment process. Monocyte chemotactic protein-3 (MCP-3), also known as CCL7, is a chemokine that attracts a broad spectrum of immune cells. Although secreted at lower levels than the better-understood monocyte chemotactic protein-1, MCP-3 is released after injury and regulates migration of leukocytes, thus facilitating inflammation. As neuroinflammation is a well-documented complication after ischemic stroke, we hypothesized that MCP-3 levels would be elevated after stroke. In light of the fact that age is the principle risk factor for stroke, and that a chronic pro-inflammatory milieu is associated with aging, we also hypothesized that MCP-3 would increase with age. Methods: Young (10 weeks) and aged (18 months) male C57B16 mice were subjected to transient (60 minute) middle cerebral artery occlusion (MCAO) or a sham surgery. MCP-3 protein levels in brain tissue and serum samples from these stroked mice, as well as from cohorts of young and aged naïve mice, were analyzed with an ELISA. Results: The comparisons of naïve aged (n=4, mean=0.042±0.005 pg/μg) to naïve young (n=4, mean=0.017±0.009 pg/μg) mice showed a significant increase (p&lt;.01) in MCP-3 in aged brain tissue. Additionally, when comparing aged MCAO to young MCAO (n=3, mean=0.01±0.007 pg/μg), MCP-3 was significantly elevated (p&lt;.01) in the aged group. In comparing aged MCAO (n=6, mean=0.31±0.11 pg/ug ) to aged sham (n=6, mean=0.03±0.006 pg/μg), MCP-3 was significantly elevated (p&lt;.05). No significant differences in MCP-3 levels in serum or between young stroke and young sham brain were seen. Conclusions: We demonstrated that levels of MCP-3 are increased post-stroke in aged mice, but not in young mice. Given what is known about the role of MCP-3 in immune cell trafficking, our data imply that MCP-3 plays a role in inflammation post-stroke, and that it also primes the aged brain for a greater inflammatory response post-stroke.