Retro-orbital Bleeding Research Articles

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

Physiological Significance of Macula Densa Neuronal Nitric Oxide Synthase-1 in Renin Release and Renin-dependent Blood Pressure Maintenance

The role of macula densa nitric oxide synthase 1 (NOS1) in regulation of renin release is controversial. This study was conducted to further elucidate the role of macula densa NOS1 in renin release and blood pressure regulation in response to salt challenges and hemorrhagic shock.Tissue specific macula densa-NOS1 knockout (KO) and wild type (WT) mice were given low (0.1% NaCl) and high (1.4% NaCl) sodium diets or induced hemorrhagic shock by retro-orbital bleeding of 0.4 ml blood. Mean arterial pressure (MAP) was measured by radio-telemetry system. Plasma renin concentration (PRC) was determined by radioimmunoassay.PRCs were 496 ± 74 and 552 ± 68 ng/ml/hr in WT and KO mice fed a normal sodium diet, respectively. Low salt intake stimulated the renin release by about 220% in WT mice (PRC = 1364 ± 217 ng/ml/hr) compared to the level of normal salt diet, however, the stimulation was significantly blunted in KO mice (PRC = 688 ± 104 ng/ml/hr). High salt intake suppressed the PRC to the level of about 61% of PRC under normal salt diet. Deletion of macula densa-NOS1 further inhibited renin release to 43% of the levels of normal salt diet.Hemorrhagic shock induced about a 3-fold increase in PRC in WT mice, while only about 54% increase in the KO mice. The MAP values were significantly higher in WT mice than in KO mice during the first 6 hours following hemorrhagic shock. Thus, WT mice showed much quicker recovery in MAP than KO mice.Our study demonstrated that macula densa NOS1 plays an important role in mediating renin release. This mechanism is essential in maintaining blood pressure under hypovolemic situations such as hemorrhagic shock. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Macula Densa Nitric Oxide Synthase 1 Controls Renin Release and Renin-Dependent Blood Pressure Changes.

The function of macula densa nitric oxide synthase 1 (NOS1) in the regulation of renin release is controversial. This study was conducted to further elucidate the role of macula densa NOS1 in renin release and blood pressure regulation in response to salt challenges and hemorrhagic shock. To investigate the specific role of NOS1 in the macula densa within the kidney in response to varying sodium concentrations in the diet, tissue macula densa-specific NOS1 knockout (MD-NOS1KO) and wild type (WT) mice were subjected to sequential low (0.1% NaCl) and high (1.4% NaCl) sodium diets. Separate groups of mice, consisting of both MD-NOS1KO subgroup and WT subgroup, were induced hemorrhagic shock by retro-orbital bleeding of 12 mL blood/kg body weight. Mean arterial pressure (MAP) was measured by a radio-telemetry system. Plasma renin concentration (PRC) was measured with the radioimmunoassay for both sodium diet and hemorrhagic shock experiments. PRCs were 371 ± 95 and 411 ± 68 ng/mL/hr in WT and MD-NOS1KO mice fed a normal sodium diet, respectively. Low salt intake stimulated an increase in the renin release by about 260% in WT mice (PRC = 1364 ± 217 ng/mL/hr, p < 0.0001) compared to the PRC under normal salt diet. However, the stimulation was significantly blunted in MD-NOS1KO mice (PRC = 678 ± 104 ng/mL/hr, p < 0.001). High salt intake suppressed the PRC to about 61% of the PRC level under a normal salt diet (p < 0.0001). Deletion of macula densa NOS1 further inhibited renin release to 33% of the levels of a normal salt diet. Hemorrhagic shock induced about a 3-fold increase in PRC in WT mice, but only about a 54% increase in the MD-NOS1KO mice (p < 0.0001). The MAP values were substantially greater in WT mice than in MD-NOS1KO mice within the first 6 hours following hemorrhagic shock (p < 0.001). Thus, WT mice showed a much quicker recovery in MAP than MD-NOS1KO mice. Our study demonstrated that macula densa NOS1 plays an important role in mediating renin release. This mechanism is essential in maintaining blood pressure under hypovolemic situations such as hemorrhagic shock.

Preclinical In Vivo Activity of the Menin Inhibitor Ziftomenib (KO-539) in Pediatric KMT2A-Rearranged Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A-rearranged (R) B-acute lymphoblastic leukemia (B-ALL), who have extremely poor clinical outcomes with event-free survival <20% for highest-risk patients. KMT2A (MLL1) is a histone lysine N-methyltransferase involved in HOX-family and MEIS1 gene regulation that is commonly rearranged with other gene partners to form oncogenic KMT2A fusion proteins that drive acute lymphoid or myeloid leukemogenesis. Recent preclinical and early clinical studies have reported successful targeting of the menin scaffold protein within KMT2A fusion complexes in adults with KMT2A-R acute leukemias (Wang ASH 2020 abstract #115, Stein ASH 2021 abstract #699), but minimal data in pediatric patients exist to date. We hypothesized that the selective menin inhibitor ziftomenib (KO-539) would have potent activity in preclinical patient-derived xenograft (PDX) models of infant and non-infant pediatric KMT2A-R ALL and could enhance chemosensitivity in these often-chemoresistant leukemias. Methods: To test these hypotheses, we first measured the ability of ziftomenib to inhibit in vitro viability and KMT2A-menin complex protein targets in human KMT2A-R and KMT2A wild-type ALL cell lines in vitro using Cell Titer Glo and immunoblotting assays, respectively. We then assessed the ability of ziftomenib as monotherapy or in combination with chemotherapy to inhibit in vivo leukemia proliferation in PDX models of de novo or relapsed KMT2A-R ALL harboring various fusion partners (n=8; Loftus Haematologica 2021) after drug dose-finding in a bioluminescent KMT2A-R ALL cell line xenograft model. NSG mice engrafted with human KMT2A-R ALL cells were randomized to treatment with vehicle, ziftomenib 150 mg/kg via oral gavage (PO) daily x 5 days/week, vincristine 1 mg/kg intraperitoneally once weekly, dexamethasone 1 mg/kg PO daily x 5 days/week, ziftomenib + vincristine, or ziftomenib + vincristine for 3-6 weeks depending on rate of ALL progression in control animals necessitating study termination. Human CD45+ CD19+ ALL cell counts were measured weekly via retro-orbital venous bleeding and in end-study murine spleens via quantitative flow cytometry analysis as previously described (Tasian Blood 2017a, Hurtz JCI 2020). Results: We observed preferential in vitro sensitivity to ziftomenib of KMT2A-R ALL cell lines HB11;19 (half-maximal inhibitory concentration [IC50] 11.1 nM), KOPN-8 (IC50 49.1 nM), and SEM (IC50 32.6 nM) versus KMT2A wild-type NALM-6 cells (IC50 400.8 nM). Ziftomenib treatment of infant, non-infant pediatric, and young adult KMT2A-R ALL PDX models (comprised of more common KMT2A-AFF1 and KMT2A-MLLT1 rearrangements or other less common KMT2A fusions) induced significant inhibition of in vivo leukemia proliferation compared to vehicle treatment. Despite the remarkable potency of single-agent ziftomenib in all models tested, combination of ziftomenib with vincristine or dexamethasone further enhanced reduction of human ALL disease burden in end-study murine spleens, even in aggressive relapsed infant ALL PDX models (representative data in Figure 1). Conclusions: Our data demonstrate in vitro target inhibition and remarkable in vivo activity of the menin inhibitor ziftomenib against infant, pediatric, and young adult KMT2A-R ALL. Ziftomenib monotherapy activity was further augmented in most models when combined with conventional chemotherapy drugs vincristine or dexamethasone, which were well-tolerated in PDX mice. Based upon early clinical safety/tolerability data for ziftomenib in adult patients with acute leukemias and our pediatric-specific data described herein, a phase 1 clinical trial of ziftomenib in combination with multi-agent chemotherapy for children with relapsed/refractory KMT2A-rearranged, NUP98-rearranged, or NPM1-mutant acute leukemias is planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of Hypoxia Inducible Factor Prolyl Hydroxylase By FG-4592 (Roxadustat) Increases Erythropoietic Stimulation in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is a devastating monogenic disorder caused by a mutation in the β-globin gene, leading to production of mutant hemoglobin S (HbS). Abnormal polymerization of deoxygenated HbS causes sickling of red blood cells (RBCs), resulting in painful vaso-occlusive crises and chronic hemolytic anemia. Etiologies of chronic anemia in SCD are multifactorial, including intravascular and extravascular hemolysis, inadequate erythropoietin (EPO) production, and abnormal iron homeostasis (Xu and Thein, Blood 2022). Hydroxyurea (HU) is the standard-of-care drug therapy for SCD and primarily reduces HbS polymerization through induction of fetal Hb (HbF) expression. However, it only modestly improves anemia and does not address other causes of anemia in SCD. Additionally, lower Hb levels in SCD are associated with chronic kidney disease (CKD), stroke, and pulmonary hypertension, arguing for the need for additional therapies targeting anemia in SCD. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a class of drugs developed for treatment of anemia in CKD and end-stage renal disease. HIF-PHIs stimulate erythropoiesis by inhibiting prolyl hydroxylase and von Hippel-Lindau (VHL)-mediated degradation of HIF-α, leading to increased hypoxic signaling and promotion of HIF-induced EPO expression in the kidneys and liver. Stimulation of erythropoiesis also increases production of erythroferrone, the main regulator of hepcidin, and suppression of hepcidin levels by HIF-PHIs may improve iron mobilization. Furthermore, HIF-PHIs can induce HbF expression in erythroblasts derived from CD34+ hematopoietic stem and progenitor cells (HSPCs) (Hsieh et al.,Blood 2007, Feng et al., ASH abstract 2021), suggesting multiple potential therapeutic benefits in SCD. Therefore, we characterized the effect of the HIF-PHI FG-4592 (roxadustat), on HbF induction in CD34+ HSPCs and on hematologic markers, EPO and hepcidin expression in a mouse model of SCD. CD34+ HSPCs from 3 donors were differentiated in vitro to erythroblasts with vehicle, 20 μM of FG-4592, or 50 μM of HU. Flow cytometric analysis on Day 9 showed that FG-4592 treatment increased % F-cells by 1.47-fold (±0.45), compared to a 2.21-fold (±0.89) increase with HU (Fig. 1). We then transplanted lethally irradiated (1000 rads) male 6-8-week-old C57BL/6J mice with bone marrow from transgenic sickle cell mice (Hb SS; Townes model, Jackson Laboratory). Twelve weeks after transplantation, mice with >80% engraftment of the donor HbSS phenotype, determined by Hb electrophoresis, were used for subsequent experiments. Vehicle, 25 mg/kg FG-4592, or 50 mg/kg FG-4592 was administered three times weekly through intraperitoneal injection for 8 weeks. All mice underwent retro-orbital bleeding at baseline (48 h pretreatment), 2, 4, 6, and 8 weeks of treatment for hematological evaluation, including Hb, hematocrit, and reticulocyte % (Fig. 1). At the end of 8 weeks, livers, kidneys, and plasma were collected. Hb and hematocrit levels increased in the 50 mg/kg FG-4592 group compared to the other groups at 4 weeks but decreased by 8 weeks for all groups. Reticulocyte % also decreased in all groups at the 4- and 8-week timepoints. Red cell distribution width (RDW) and platelet count up-trended while mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) down-trended over the 8 weeks, suggestive of an evolving iron deficiency contributing to worsening anemia. Quantitative PCR detected a 182% increase in liver EPO transcript and a 68% increase in kidney EPO transcript in the 50 mg/kg FG-4592 treated group, compared to vehicle. No changes in liver hepcidin transcription were observed in any group. ELISA analysis detected increased plasma EPO and decreased plasma hepcidin levels with increasing doses of FG-4592. None of these trends, except increases in RDW, were statistically significant. Our study is the first to characterize the effect of HIF-PHI drugs in a mouse model of SCD. Preliminary data confirm an effect of FG-4592 on HbF induction and suggest that FG-4592 may increase EPO expression and decrease hepcidin levels in SCD mice. These results highlight the potential of prolyl hydroxylase inhibition as a novel therapeutic approach for chronic anemia in SCD. Future directions include testing higher doses of FG-4592 to induce a stronger erythropoietic effect and monitoring additional markers of iron homeostasis in our mouse model of SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tualang Honey Supplementation Alleviates Obesity and Dyslipidaemia in High Cholesterol Diet Induced Non-Alcoholic Steatohepatitis Animal Model

INTRODUCTION: Dyslipidaemia and obesity are two main features of non-alcoholic steatohepatitis (NASH). This study aimed to investigate the effects of Tualang honey (TH) supplementation on bodyweight, liver weight, and lipid profiles in high cholesterol diet (HCD) induced NASH animal model. MATERIALS AND METHODS: Sixteen Sprague-Dawley rats were given 12% HCD for 16 weeks to induce NASH. These animals were divided into 4 groups; Group 1 (continued HCD), Group 2 (changed to normal diet), Group 3 (normal diet and TH 1.2g/kg) and Group 4 (normal diet and TH 3.0g/kg) for the following 4 weeks. Bodyweight was measured daily. At the end of the study, blood was collected via retro-orbital bleeding and the rats were sacrificed to harvest their liver. RESULTS: The group 4 rats had significantly lower mean final bodyweight than rats in group 1, 2 and 3 (478.0±24.4 vs. 641.5±25.1, 593.8 ±29.3, 552.0±72.9 g, p&lt;0.05). Animals in group 4 were also found to have a significantly lower mean liver weight compared to groups 1 and 2 (12.9±0.9 vs 20.1±2.2, 15.7±1.2 g, p&lt;0.05). In comparison to controls, the mean concentration of total cholesterol was significantly lower in all the other groups and the lowest mean concentration of triglycerides was recorded in group 4 with significant difference when compared to the controls (0.9±0.4 vs 3.6±0.4 mmol/L, p&lt;0.05). CONCLUSION: The change from HCD to a normal diet coupled with TH supplementation has been shown to reduce bodyweight, liver weight, total cholesterol and triglyceride levels in the 12% HCD NASH induced animal models.

Role of 12-LOX in the Platelet Storage Lesion

Background: 12-lipoxygenase (12-LOX) is an enzyme abundant in platelets which can contribute to the platelet storage lesion by oxidizing polyunsaturated fatty acids (PUFAs) released from phospholipid membranes. We and others have shown that the PUFA arachidonic acid (AA) and its lipid oxidation products, such as 12-hydroxyeicosatetraenoic acid (12-HETE), accumulate during storage and have inhibitory effects on platelet recovery, survival, and function. However, several PUFAs are substrates for 12-LOX, and their resulting oxylipins may have different effects. We used targeted metabolomics to quantify PUFAs and oxylipins and platelet function assays to characterize function of fresh and stored wild-type (WT) and 12-LOX -/- platelets.Methods: Blood from WT and 12-LOX -/- mice was collected by retro-orbital bleeding. Platelet-rich plasma (PRP) was generated from whole blood. After fresh samples were aliquoted, the remaining PRP was separated in two groups. One group was stored at room temperature with agitation (RT) for 24 hours, and the other for 48 hours. Metabolites were extracted from samples and quantified by targeted metabolomics as described previously. We assessed platelet function by αIIbβ3 integrin activation by flow cytometry. In vivo recovery of function was measured by transfusing stored platelets into UBiC-GFP mice and stimulating platelets with agonists, followed by gating for transfused (GFP-negative) platelets by flow cytometry. For recovery and survival, we traced biotinylated fresh, 24h, or 48h-stored platelets after transfusion in vivo.Results: We quantified metabolites present in platelets by targeted metabolomics to monitor their changes in concentration over storage time. Among the 10 PUFAs and 28 related oxylipins we analyzed, 15 of 38 analytes showed a significant difference in PRP from WT and 12-LOX-/- mouse samples. The major metabolites of 12-LOX include 12-HETE, 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA), from AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). 12-HETE, 12-HEPE, and 14-HDHA were only detected at <8 nmol/L levels in fresh PRP from 12-LOX -/- mice compared to 668 ± 409nM, 149 ± 85nM, and 295 ± 154nM from WT mice, respectively. After 24 hours of storage at RT, 12-HETE, 12-HEPE, and 14-HDHA dramatically increased to 29.0±4.2µM, 3.7±1.1µM, and 6.3±0.8µM in PRP from WT mice, respectively. As expected, these same metabolites remained at low nmol/L levels in 12-LOX-/- samples during storage accompanied by a significant increase of their precursors AA, EPA, and DHA due to lack of 12-LOX activity. Interestingly, there was also a significant reduction in 15-HETE, 17-HDHA, and 13-hydroxyoctadecadienoic acid (13-HODE) in the 12-LOX -/- mice compared to the WT mice, which are primarily produced by the 15-LOX enzyme. Additionally, we observed a significant decrease of metabolites mediated via the cyclooxygenase (COX) pathway in PRP from 12-LOX-/- mice, including prostaglandin E2 (PGE2), PGD2, thromboxane B2, and 12-hydroxyheptadecatrienoic acid (12-HHTrE). Function-wise, fresh 12-LOX -/- platelets were less responsive to agonists compared to WT platelets. Surprisingly, after transfusion of fresh 12-LOX -/- platelets, we found comparable αIIbβ3-integrin activation results after 1, 4, and 24h of circulation time. In contrast, 24h and 48h of storage of 12-LOX -/- platelets led to significantly lower pre-activation at baseline and a significantly lower activation response than WT platelets after 1h and 4h of circulation time. No significant differences were observed after 24h of circulation time. We observed a clear trend for longer survival after 24 and 48h of storage.Conclusions: We found many metabolic changes between 12-LOX -/- and WT mice during storage. While the 12-LOX -/- mouse model highlights the primary metabolic differences that occur without 12-LOX activity, other changes, such as differences in COX or additional LOX isoform activity, may attenuate oxylipin production. Functionally, we observed less pre-activation and better survival in functional studies, but this may be due to a combined effect of each of these individual metabolites. Future studies will have to determine the roles of individual oxylipins. DisclosuresStolla: Cerus: Research Funding.

Development and Validation of [18f](2 S,4 R)-4-Fluoroglutamine in Multiple Myeloma Mouse Models

Glutamine (Gln) addiction has been recently described as a typical metabolic feature of MM by our group. In order to sustain high Gln demand, MM cells upregulate the expression of the Gln transporters ASCT2, SNAT1 and LAT1 and are characterized by fast Gln uptake. Currently, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) is the standard tracer for the positron emission tomography/computed tomography (PET/CT) scan to detect both medullary and extramedullary disease in MM patients. However, FDG-PET has some limitations, since there is a portion of MM patients who are false-negative. For these reasons, the aim of our study is to characterize Gln metabolism in vivo in different MM mouse models and investigate the possible use of Gln as a PET tracer in MM.To this purpose, we have firstly synthesized enantiopure (2 S,4 R)-4-fluoroglutamine (4-FGln) and validated it in human MM cell lines (RPMI8226 and JJN3) comparing its uptake with that of 3H-labelled Gln. Both Gln and 4-FGln were actively accumulated by MM cells. Inhibition analysis revealed that ASCT2 was the major entry route of both compounds.We then carried out the radiosynthesis of [18F]4-FGln and tested its uptake for MM detection by PET in comparison with [18F]FDG. Firstly, we characterized [18F]4-FGln kinetics using immunodeficient NOD/SCID mice subcutaneously injected with JJN3. In this xenograft model, [18F]FDG- and [18F]4-FGln-PET were performed after plasmacytomas became palpable. In tumor, [18F]4-FGln uptake peaked at 25 min slowly declining thereafter. The Logan plot confirmed linearity starting at 15 min from injection, consistent with largely reversible tracer exchange.Thereafter, [18F]FDG and [18F]4-FGln distribution were assessed in both syngeneic (murine Vk12598 cells) and xenograft ( human JJN3 cells) MM models.C57BL/6 mice were injected intravenously with Vk12598 cells obtained from transgenic Vk*MYC mice. Mice were monitored weekly for M-spike and MM progression by retro-orbital bleeding. At weeks 3, 4 and 5, mice underwent PET with [18F]FDG and [18F]4-FGln on consecutive days. The expression of ASCT2, checked in the femur of Vk12598 MM bearing mice, increased along with MM progression. Uptake of both tracers was measurable in the spleen of MM mice by week 3, reached a peak at week 4 and declined thereafter. In JJN3 model, all the tumors were positive for [18F]FDG and displayed [18F]4-FGln uptake (18F]4-FGln: tumor to muscle ratios (T/M): 1.6 ± 0.1; [18F]FDG: T/M: 3.5 ± 1.1). Tumor volume (896.8 ±349.00 mm3), [18F]4-FGln uptake and [18F]FDG uptake (T/M: 2.3 ± 0.3 and 7.1 ± 2.6, respectively) increased after 1 week ([18F]4-FGln p = 0.0156; [18F]FDG p = 0.0167).Thereafter, the effect of bortezomib (BOR) was investigated to evaluate the potential use of [18F]4-FGln to monitor anti-MM treatment. JJN3-bearing mice were treated with BOR (1mg/kg) by intravenous injections twice weekly. Before and after treatment, animals performed the PET acquisitions with [18F]FDG and [18F]4-FGln on consecutive days and were sacrificed for post-mortem analysis on day 7. As expected, bortezomib treatment reduced tumor size compared with vehicles (172.0 ± 86.6 vs 687.9 ± 286.5 mm3; p = 0.0006).PET analysis performed after BOR treatment showed that BOR significantly reduced the uptake of both radiopharmaceuticals in comparison with vehicles (p< 0.05). BOR-treated mice were classified as responders and non responders according to the adapted RECIST score. Interestingly, responder mice showed a reduction of [18F]4-FGln T/M ratio and [18F]4-FGln-related tumor volume. On the contrary, all mice displayed increased [18F]FDG parameters independently from the response. Lastly, tracer overlap analysis showed that the area of relative exclusive uptake of [18F]4-FGln decreased in responders compared to non responders (9% versus 32%, respectively, p=0.04), while that of [18F]FDG increased in responders compared to non responders (82% versus 23%, respectively, p=0.04).Our data indicate that [18F](2 S,4 R)-4-FGln is a new PET tracer in pre-clinical MM models, providing the rationale to design studies in MM patients. DisclosuresGiuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: congress, Research Funding; Millenium Pharmaceutical: Other: clincial studies; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical studies, congress, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Other: clinical studies; Bristol Mayers Squibb: Other: congress.

Impact of environmental conditions on the levels of stress and breeding performance in Wistar rats: conventional environment versus environmentally controlled housing.

It has been established that providing comparable and standard environmental conditions to house experimental rats is of high importance. However, there is growing evidence that rats prefer higher temperatures owing to their thermoneutral zone that lies between 28-34°C. This experiment validates the stress levels of Wistar rats of both sexes housed in conventional conditions about 24-31°C and a relative humidity of 70-92% in a tropical animal facility when compared to rats housed in controlled temperatures of 20-24°C with humidity maintained between 30-70%. Adequate air-changes to maintain ammonia-free environment was provided with a power-exhaust system in a conventional setup and with the individually ventilated caging system in the environmentally controlled group. To assess stress, faecal corticosteroid metabolite assay was done in naïve animals and in a set of animals that underwent retro-orbital bleeding under general anaesthesia. Mothering ability, fecundity and preweaning mortality were also compared between animals housed in two different conditions. Results showed no differences in stress hormone levels between the groups. However, faecal weights differed between males and females in both naïve and orbital bled groups. Animals housed in controlled environment clearly had better breeding data with lesser preweaning mortality in comparison to the conventionally housed rats. Further studies are required to elucidate whether the results are comparable when conducted during all seasons of the year at different locations across the globe.

Biological and toxicological evaluation of Rhus trilobata Nutt. (Anacardiaceae) used traditionally in mexico against cancer

BackgroundRhus trilobata Nutt. (Anacardiaceae) (RHTR) is a plant of Mexico that is traditionally used as an alternative treatment for several types of cancer. However, the phytochemical composition and potential toxicity of this plant have not been evaluated to support its therapeutic use. Therefore, this study aimed to evaluate the biological activity of RHTR against colorectal adenocarcinoma cells, determine its possible acute toxicity, and analyze its phytochemical composition.MethodsThe traditional preparation was performed by decoction of stems in distilled water (aqueous extract, AE), and flavonoids were concentrated with C18-cartridges and ethyl acetate (flavonoid fraction, FF). The biological activity was evaluated by MTT viability curves and the TUNEL assay in colorectal adenocarcinoma (CACO-2), ovarian epithelium (CHO-K1) and lung/bronchus epithelium (BEAS-2B) cells. The toxicological effect was determined in female BALB/c mice after 24 h and 14 days of intraperitoneal administration of 200 mg/kg AE and FF, respectively. Later, the animals were sacrificed for histopathological observation of organs and sera obtained by retro-orbital bleeding for biochemical marker analysis. Finally, the phytochemical characterization of AE and FF was conducted by UPLC-MSE.ResultsIn the MTT assays, AE and FF at 5 and 18 μg/mL decreased the viability of CACO-2 cells compared with cells treated with vehicle or normal cells (p ≤ 0.05, ANOVA), with changes in cell morphology and the induction of apoptosis. Anatomical and histological analysis of organs did not reveal important pathological lesions at the time of assessment. Additionally, biochemical markers remained normal and showed no differences from those of the control group after 24 h and 14 days of treatment (p ≤ 0.05, ANOVA). Finally, UPLC-MSE analysis revealed 173 compounds in AE-RHTR, primarily flavonoids, fatty acids and phenolic acids. The most abundant compounds in AE and FF were quercetin and myricetin derivates (glycosides), methyl gallate, epigallocatechin-3-cinnamate, β-PGG, fisetin and margaric acid, which might be related to the anticancer properties of RHTR.ConclusionRHTR exhibits biological activity against cancer cells and does not present adverse toxicological effects during its in vivo administration, supporting its traditional use.

A study on the protective effects of CpG oligodeoxynucleotide-induced mucosal immunity against lung injury in a mouse acute respiratory distress syndrome model.

This study aims to determine the feasibility of using oligodeoxynucleotides with unmethylated cytosine-guanine dinucleotide sequences (CpG ODN) as an immunity protection strategy for a mouse model of acute respiratory distress syndrome (ARDS). This is a prospective laboratory animal investigation. Twenty-week-old BALB/c mice in Animal research laboratory were randomized into groups. An ARDS model was induced in mice using lipopolysaccharides (LPSs). CpG ODN was intranasally and transrectally immunized before or after the 3rd and 7th days of establishing the ARDS model. Mice were euthanized on Day 7 after the second immunization. Then, retroorbital bleeding was carried out and the chest was rapidly opened to collect the trachea and tissues from both lungs for testing. CpG ODN significantly improved the pathologic impairment in mice lung, especially after the intranasal administration of 50 μg. This resulted in the least severe lung tissue injury. Furthermore, interleukin-6 (IL-6) and IL-8 concentrations were lower, which was second to mice treated with the rectal administration of 20 µg CpG ODN. In contrast, the nasal and rectal administration of CpG ODN in BALB/c mice before LPS immunization did not appear to exhibit any significant protective effects. The intranasal administration of CpG ODN may bea potential treatment approach to ARDS. More studies are needed to further determine the protective mechanism of CpG ODN.

Targeting the Second Bromodomain (BDII) of BET Family with Abbv-744 Results in Robust Anti-Leukemia Activity in AML Models with Excellent In Vivo Tolerability

The BET family proteins consist of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two highly conserved bromodomains (BDI and BDII). First generation BET inhibitors, including ABBV-075, are pan-BET inhibitors that bind with nearly equimolar affinity to BDI and BDII. Pan-BET inhibitors generally block transcription and inhibit the proliferation of a large spectrum of tumor types, but exhibit a narrow therapeutic index. ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia activities of ABBV-744 in acute myelogenous leukemia (AML) cell lines and primary samples.In AML cell lines, ABBV-744 induced G1 cell cycle arrest and apoptosis with caspase-3 activation and PAPR cleavage through regulation of the key proteins that are involved in cell cycle and apoptosis pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc). Mechanistically, ABBV-744 displaced BRD4 from the regulatory region of BCL2, RUNX1, BCL2L1 and IL8 genes by ChiP-Seq. MLL-rearranged cells were most sensitive to BDII inhibition, and ABBV-744 displaced both, BRD4 and MLL from the regulatory regions of BCL2 and c-Myc genes in MV4;11 (MLL-AF4) and Nomo-1 (MLL-AF9) cells. We examined anti-leukemia activity of ABBV-744 in 12 primary AML samples with diverse genetic alterations. ABBV-744 inhibited cell growth in 8/12 primary samples (20.2%-77.9%) and induced apoptosis (26.7 ± 9.6% in ABBV-744 group vs 14.1± 3.9% in DMSO group, p=0.007, Fig 1A). We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to ABBV-744 tend to have higher pro-apoptosis (TXNIP, DR5) and lower anti-apoptosis (BCL2, EXPO) than resistant samples.For efficacy assessment in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, IDH1 and NPM1 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle or single agent ABBV-744 at a well-below MTD dose of 9.4 mg/kg for 21 days. The median survival time of mice treated with ABBV-744 (Median survival: 76 days) was significantly higher compared to untreated mice (Median survival: 67.5 days, p=0.007, Fig 1B). Next, the anti-leukemia efficacy of ABBV-744 was tested in 7 additional AML PDX models. NSG mice were injected with 7 AML PDX (one mouse per cohort design). After engraftment was confirmed by retro-orbital bleeding, mice were treated with vehicle or ABBV-744. Circulating tumor burden was measured by flow cytometry in peripheral blood samples from mice collected on indicated days of therapy. In five of the 7 models, ABBV-744 treatment delayed AML progression compared to untreated mice (survival days: 135 vs 105, 205 vs 153, 62 vs 46, 118 vs 77, 97 vs 86). No significant impact on mice' weight was noted (Fig 1C), and no clinical signs of toxicity recorded over the course of therapy.In summary, blockade of BET family bromodomain BDII promotes apoptotic cell death and suppresses proliferation in the majority of primary AML cells. Importantly, ABBV-744 therapy reduced AML tumor burden and extended survival in AML PDX models at a well-below MTD dose. Together, these results provide proof of concept that highly selective inhibitors of the second bromodomain of BET family may maintain robust anti-leukemia efficacy in AML while exhibiting improved tolerability relative to pan-BET inhibitors. [Display omitted] DisclosuresUziel:AbbVie Inc.: Employment. Shen:AbbVie Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding. Lin:AbbVie Inc: Employment. Lu:AbbVie Inc: Employment. Zhang:AbbVie Inc: Employment.

High-Level Setd8 Expression Is Important for Establishment of Normal Patterns of Erythroid Gene Expression and Is Essential for Stress Erythropoiesis

Setd8 is the sole histone methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 is expressed at basal levels in most cell types and is important for many basic cellular functions, including cell cycle progression, transcriptional regulation, and mitotic chromatin condensation. Setd8 is expressed ~10-fold higher in erythroblasts than any other cell type and during erythroid maturation of human CD34+ HSPC, Setd8 protein levels increase in parallel with Gata1 levels, suggesting that Setd8 may have an erythroid-specific function(s). Consistent with this hypothesis, erythroid-specific deletion of Setd8 was embryonic lethal, resulting in profound anemia. Setd8-null erythroblasts had cell cycle abnormalities, failure of transcriptional repression, and defective terminal erythroid maturation. (Malik et al., Cell Reports, 2017). These studies provided important insights into the function of Setd8 in erythroid cells, but were not able to clearly delineate the “housekeeping” functions of Setd8 from its specific functions in erythropoiesis.To identify the erythroid-specific functions of Setd8, we sought to identify and disrupt the enhancer that drives high level Setd8 expression in erythroid cells. Using publically available ChIP-seq data sets, we identified a putative enhancer located in intron 1 of the SETD8 gene that was occupied by Gata1, Tal1, and H3K4me1 in human erythroblasts derived from culture of CD43+ HSPCs. This putative enhancer was able to drive luciferase expression in a reporter gene assay, and deletion of the Gata1:Tal1 site at the center of this region was sufficient to abrogate reporter gene activity. Based on these data, we hypothesized that this was the enhancer that drives high level expression of Setd8 in erythroid cells. To test this hypothesis, we used CRISPR/Cas9 genome editing to delete this region in HUDEP-2 cells. Briefly, Cas9 and guide RNA ribonucleoprotein complexes targeting the enhancer were delivered into the cells using electroporation (Gundry et al., Cell Reports, 2015). PCR and sequencing were used to confirm genome editing in monoclonal cell lines. Homozygous deletion of the enhancer (Δ/Δ) reduced SETD8 expression to 27.8% of WT (+/+) controls by RT-qPCR (n=3 for each genotype; p=0.0018). Decreased Setd8 protein levels and H4K20 mono-methylation was confirmed by Western blot. Further supporting an important function of Setd8 in erythropoiesis, deletion of the enhancer and exon 7 in CD34+ HSPCs resulted in a decreased efficiency of erythroid colony formation to 49.6% of control (n=5, p=0.0359).To gain insights into Setd8 gene regulation in erythroid cells, we performed RNA-seq, comparing the Δ/Δ and +/+ enhancer lines. In total, there were 603 genes differentially expressed (p<0.05; fold change >1.5), including SETD8, FAS, and CDKN1A (p21Cip1). Pathway analyses identified numerous genes associated with apoptosis and cell death to be up-regulated. Intriguingly, multiple genes in important for stress erythropoiesis were differentially expressed in the Setd8 Δ/Δ and +/+ enhancer lines and were also differentially expressed in Setd8-null murine erythroblasts (Malik et al., Cell Reports, 2017). Most notably, both the Δ/Δ enhancer lines and the Setd8-null erythroblasts had significantly higher levels of Fas death receptor transcript than control cells. Down-regulation of Fas is essential for stress erythropoiesis (Liu et al., Blood, 2006). We therefore hypothesized that Setd8 is important for the stress erythropoiesis response. To test this hypothesis, we subjected EpoR-Cre+/-;Setd8fl/+ (Setd8Δ/+) and EpoRCre+/-;Setd8+/+ (Setd8+/+) mice to anemic stress by retro-orbital bleeding. Setd8Δ/+ and Setd8+/+ mice had similar hematocrit after anemic stress (26.6 vs 29.4%; p=0.216), but the Setd8Δ/+ had an impaired ability to mount a stress response, with a lower MCV (43.0 vs 45.1 fL, p=0.003) and reticulocyte count (8.05 vs 2.14%, p=0.031) Consistent with the transcriptomic data, Setd8Δ/+ mice had higher levels of Fas transcript in splenic erythroblasts than Setd8+/+ controls. Together, these data suggest that high level Setd8 expression is important for normal erythroid maturation and gene expression, and for regulating the stress erythropoiesis response. DisclosuresNo relevant conflicts of interest to declare.

In Vivo Blockade of Beta-1 and Beta-2 Integrin Activity Inhibits Splenomegaly in JAK2-V617F Positive Myeloproliferative Disease

Introduction:Recently, we showed that in chronic myeloproliferative neoplasia (CMN), β1 and β2 integrins are overactivated on leukocytes in patients and in a JAK2-V617F knock-in mouse model (Gupta et al., Leukemia, 2017; Edelmann-Stephan et al., JCI, In-Press Preview) resulting in increased adhesion of leukocytes to vascular cell adhesion molecule 1 (VCAM-1) and to intercellular cell adhesion molecule 1 (ICAM-1). VCAM-1 and ICAM-1 are abundantly expressed on endothelial cells and in spleen tissue. In JAK2-V617F induced CMN, current therapeutic strategies for symptomatic splenomegaly as hydroxyurea, JAK inhibitors or allogeneic stem cell transplantation (SCT) are effective, however, often limited in duration of efficacy or in eligibility for SCT. Therefore, there is a need to deeper understand the pathophysiology of splenomegaly in CMN in order to develop novel therapeutic approaches. We sought to investigate the effects of in vivo blockade of β1 and β2 integrin overactivation on spleen size, cellular composition of spleen and peripheral blood counts.Material and Methods:11 weeks old Vav1-Cre x JAK2+/V617F mice were treated with a combination of 200µg neutralizing anti-α4β1-integrin (VLA4) antibody (BioXCell, USA) plus 200µg neutralizing anti-β2 integrin antibody (BD Pharmingen, USA) (N=6) and with isotype controls (each 200µg) (N=3), respectively. Animals were injected i.p. on day 1 and day 8. Small animal MRI was used to serially assess the development of spleen volume on days 1, 8, and 15. Abdominal MRI-images employing a slice thickness of 1-2 mm were obtained. The volume of spleens were computed according to radiology standards. Peripheral blood was collected via retro-orbital bleeding and blood counts were measured on days 1, 8, and 15. Thereafter, mice were sacrificed by cervical dislocation and spleens were removed for further analysis by FACS.Results and Discussion:The applied antibody injections were well tolerated and all animals survived the planned observation period. At the start of the experiments (day 1), splenomegaly was confirmed by MRI. As expected, the spleen volumes were similar in the cohorts of isotype and β1/β2 integrin antibody treated JAK2+/V617F mice: 550±76 mm3 (mean±SD) and 518±96 mm3, respectively (Fig. 1). At day 8, upon a single injection of β1/β2 integrin antibodies, a significant decrease in mean spleen volume from baseline 518±96 mm3 to 414±32 mm3 (p=0.0302) was observed (Fig. 1). The correspondent mean±SD for the isotype antibody treated animals was 594±63 mm3. Thus, on day 8, a 30.3% reduction in spleen volume was demonstrated upon β1/β2 integrin antibody treatment in comparison to isotype treatment (p=0.0006). On day 15, the mean spleen volumes of isotype-treated and β1/β2 integrin antibody-treated mice were 665±187 mm3 and 460±69 mm3 (p=0.0410). These results demonstrate that in JAK2-V617F induced disease, blocking of overactivated β1/β2 integrins reduces splenomegaly within a short period of time (8 days). Longer application schedules may build up even stronger and stable decrease in spleen size.In addition to MRI imaging, we also analyzed the composition of peripheral blood cells. At the start of the experiments on day 1, blood parameters showed the typical abnormalities of myeloproliferative disease in both cohorts. Mean counts in isotype treated animals were: WBC = 14,300/µL, hematocrit = 80.3 %, PLT = 1283/nL. Mean counts in β1/β2 integrin antibody treated animals were: WBC = 13,850/µL, hematocrit = 76.0 %, PLT = 1348/nL. This confirms that the JAK2-V617F mouse model used indeed resembles polycythemia vera. In general, the changes observed upon β1/β2 integrin antibody treatment in blood were subtle: the WBC slightly decreased from 13,850/µL to 12,520/µL on day 15, whereas RBC, HCT and PLT remained nearly unchanged. This indicates tolerability of β1/β2 integrin antibody treatment at the level of the hematopoietic system. In isotype treated mice, peripheral blood counts also stayed stable. Data obtained by FACS analysis of peripheral blood and of spleen tissue (composition in granulocytes, B-cells and T-cells) will be presented.Overall, our results indicate a novel role of JAK2-V617F induced overactivation of β1/β2 integrins in development of splenomegaly. Thus, modulation of integrin activity may have therapeutic potential to reduce the spleen volume in CMN patients insufficiently responding to standard treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Potent effect of KISS1-54 DNA vaccine compared with KISS1-10 DNA vaccine in inhibiting the fertility of female rats

BackgroundMost studies on immunocastration currently focused on male animals. However, immunization of male animals does not completely inhibit sexual behavior and fertility. This study aimed to compare the immunocastration effect of KISS1 DNA vaccines encoding different lengths of kisspeptins in female rats for effective castration effects on both male and female rats. MethodsFifteen female rats were randomly divided into three groups. The rats in T1 group or T2 group was orally given respectively KISS1-54 or KISS1-10 DNA vaccines with fused tPA signal peptide, and the control group (Group C) was orally administered with empty vector vaccine, at a dose of 5 × 109 CFU/rat at weeks 0, 3 and 6 of the study. Blood samples were collected by retroorbital bleeding before primary immunization and at weeks 3 and 9 after primary immunization. ResultsBoth KISS1-54 and KISS1-10 DNA vaccines induced the body’s humoral immune response, and the anti-kisspeptin antibody titres in the T1 group were significantly higher than that in T2 and C groups (p < 0.05). The rats in T1 group has lower serum kisspeptin and estradiol levels than those in T2 and C groups and smaller litter size of rats than those in the control group after mating (p < 0.05). No significant difference was observed between T2 and C groups. The levels of KISS1 and GPR54 mRNA in the hypothalamus and ovaries of the T1 group were significantly lower than that in control group. However, the levels of KISS1 mRNA in the T2 group were significantly lower than that in the control group only in ovaries (p < 0.05). ConclusionThe oral KISS1-54 DNA vaccine with fused tPA signal peptide was more effective than that KISS1-10 DNA vaccine in suppressing fertility of female rats.

ATR kinase inhibitors NVP-BEZ235 and AZD6738 effectively penetrate the brain after systemic administration

Ataxia Telangiectasia and Rad3 related protein (ATR) is a central mediator of the response to DNA damage that may cause the quiescent resistance of cancer initiating cells to genotoxic radiotherapy. NVP-BEZ235 is a dual PI3K/mTOR inhibitor that also effectively targets ATR with IC50 = 21 × 10− 9 M in cells. AZD6738 does not target significantly PI3K/mTOR-related kinases but specifically inhibits ATR with IC50 = 74 × 10− 9 M in cells. Both drugs have been proposed as radiosensitizers of different tumors including glioblastoma (GB), the most malignant brain tumor. In order to study the radiosensitizing properties of ATR inhibitors NVP-BEZ235 and AZD6738 towards GB, we have preliminarily investigated their capacity to penetrate the brain after systemic administration. Tumor-free CD-1 mice were inoculated i.p. with 25 mg/Kg body weight of NVP-BEZ235 or AZD6738. 1, 2, 6 and 8 h later, blood was collected by retro-orbital bleeding after which the mice were euthanized and the brains explanted. Blood and brain samples were then extracted and NVP-BEZ235 and AZD6738 concentrations determined by High Performance Liquid Chromatography/Mass Spectrometry. We found for NVP-BEZ235 and especially for AZD6738, elevated bioavailability and effective brain penetration after intraperitoneal administration. Albeit low drug and radiation dosages were used, a trend to toxicity of NVP-BEZ235 followed by ionizing radiation (IR) towards mice bearing primary glioma initiating cells (GIC)-driven orthotopic tumors was yet observed, as compared to AZD6738 + IR and vehicle+IR. Survival was never improved with median values of 99, 86 and 101 days for vehicle+IR, NVP-BEZ235 + IR and AZD6738 + IR-treated mice, respectively. Although the present results indicate favorable pharmacokinetics properties of ATR inhibitors NVP-BEZ235 and AZD6738, they do not lend support to their use as radiosensitizers of GB.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Biochemical and histopathological changes of subacute cadmium intoxication in male rats.

Biochemical and histopathological effects of subacute intoxication of rats with cadmium (Cd) were studied in rats. Twenty adult healthy male albino rats were randomly divided into two duplicate groups (five rats in each cage); (1) control group where rats were provided with standard diet and water ad-libitum, (2) Cd group where rats were subjected to freshly prepared Cd chloride solution (CdCl2) 200mg/l in drinking water daily for 8weeks, the whole duration of experiment. Blood samples were obtained after 4weeks, via retro-orbital bleeding for separation of serum. Five rats were killed, each sacrifice by decapitation for collection of kidneys and heart. Disturbed renal and cardiac functions were achieved after 4weeks as indicated by the increase of most biochemical parameters measured in the serum, renal, and cardiac tissues. Histopathological examination of kidneys and hearts showed pathological alterations in Cd-intoxicated rats after 4 and 8weeks with hematoxylin and eosin (H&E) and Masson trichrome stains. It was concluded that subacute exposure of rats to Cd (200mg/l) in drinking water daily induced glomerular shrinkage, focal renal, and cardiac fibrosis at 4 and 8weeks.

Evaluation and Optimization of Blood Micro-Sampling Methods: Serial Sampling in a Cross-Over Design from an Individual Mouse.

Current practices applied to mouse pharmacokinetic (PK) studies often use large numbers of animals with sporadic or composite sampling that inadequately describe PK profiles. The purpose of this work was to evaluate and optimize blood microsampling techniques coupled with dried blood spot (DBS) and LC-MS/MS analysis to generate reliable PK data in mice. In addition, the feasibility of cross-over designs was assessed and recommendations are presented. The work describes a comprehensive evaluation of five blood microsampling techniques (tail clip, tail vein with needle hub, submandibular, retro-orbital, and saphenous bleeding) in CD-1 mice. The feasibility of blood sampling was evaluated based on animal observations, ease of bleeding, and ability to collect serial samples. Methotrexate, gemfibrozil and glipizide were used as test compounds and were dosed either orally or intravenously, followed by DBS collection and LC-MS/MS analysis to compare PK with various bleeding methods. Submandibular and retro-orbital methods that required non-serial blood collections did not allow for inter-animal variability assessments and resulted in poorly described absorption and distribution kinetics. The submandibular and tail vein with needle-hub methods were the least favorable from a technical feasibility perspective. Serial bleeding was possible with cannulated animals or saphenous bleeding in non-cannulated animals. Of the methods that allowed serial sampling, the saphenous method when executed as described in this report, was most practical, reproducible and provided for assessment of inter-animal variability. It enabled the collection of complete exposure profiles from a single mouse and the conduct of an intravenous/oral cross-over study design. This methodology can be used routinely, it promotes the 3Rs principles by achieving reductions in the number of animals used, decreased restraints and animal stress, and improved the quality of data obtained in mouse PK studies.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

New Therapeutic Approach for Central Nervous System Lymphoma By Intracerebroventricular Delivery of CD19CAR T Cells

Central nervous system lymphoma (CNSL) is a lymphoid malignancy in which tumors from lymph tissue start in the brain, spinal cord, eye, and/or meninges (primary CNSL) or present as a result of metastasis from initial systemic sites to the CNS (secondary CNSL). The most common CNS lymphomas (about 90%) are B-cell lymphomas. The incidence of primary CNS lymphoma has been increasing over the past 20 years. Multifocal lesions are common. CNS lymphomas carry a worse prognosis than systemic lymphoma. Only a few chemotherapeutic drugs can cross and achieve a therapeutic concentration in the CNS. Therefore, effective treatment is limited and the outcome of disease in relapsed or refractory setting is poor.Recent studies show that intraventricular delivery of rituximab in CNS lymphomas is well tolerated. T cell products that are genetically engineered with chimeric antigen receptors (CARs) targeting CD19 have broad application for adoptive therapy of B cell lineage malignancies and have shown tremendous potential in treatment of systemic lymphoma. In all CD19CAR T cell trials, T cell products are administrated intravenously. CD19CAR T cell trafficking in cerebrospinal fluid (CSF) is frequently reported but most if not all protocols exclude patients with active CNS involvement. In this study, we set out to investigate the feasibility and efficacy of the use of CD19CAR T cells to treat CNSL.Methods and Results: Isolated naïve and central memory T cells (Tn/Tmem) were genetically modified with CD19CAR lentivirus and expanded in vitro for 14 days. 0.1x10^6 human B cell lymphoma Daudi cells were injected intracranially into NSG mice. Tumor was allowed to engraft for 5 days. We administered CD19CAR T cells via three different delivery routes: intracranial local infusion with 1x10^6 CD19CAR T cells (i.c), intracerebroventricular (i.c.v) administration with 1x10^6 cells to bypass the blood-brain barrier and target tumor throughout the entire CNS, and intravenous injection (i.v) with 3x10^6 cells. We repeatedly observed in 2 separate experiments (N=5 mice in each experiment) that both a single i.c infusion and a single i.c.v delivery of CD19CAR T cells were able to completely eradicated CNS lymphoma in all mice by day 14 post CAR T cell infusion; and that a single dose of i.v infusion induced significant anti-CNSL activity with a slightly delayed response as compared to i.c and i.c.v treatment and all mice achieved complete remission 21 days post T cell infusion. CAR T cells were detected in peripheral blood obtained from retro-orbital bleeding, not only in the i.v treated mice, but also in i.c.v treated mice 28 days after CAR T cell infusion, suggesting that i.c.v not only controls CNSL but may also play a role in immune surveillance for systemic tumors. To confirm this, we established an NSG CNS B cell lymphoma model by also inoculating subcutaneous tumors on the animal’s flank, 3 weeks prior to i.c tumor injection into the same mouse. CD19CAR T cells were delivered via i.c.v 5 days after i.c. tumor injection. CAR T cell injection resulted in complete remission of both the brain tumor and the flank tumor 14 days after CAR T cell administration.In conclusion,intracerebroventricular delivery of CD19CAR T cells is a promising and feasible therapeutic approach for both primary central nervous system lymphoma and systemic lymphoma with concurrent CNS involvement. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.