Mouse Peripheral Blood Cells Research Articles

Evaluation of cytotoxicity, acute toxicity, genotoxicity and antioxidant and antigenotoxicity activities of the sarcotesta fraction of punica granatum L. rich in lectin (PgTel)

ABSTRACT Punica granatum, popularly known as pomegranate, is a fruit tree with wide worldwide distribution, containing numerous phytochemicals of great medicinal value. The aim of the present study was to determine the phytochemical profile and antioxidant potential of a protein fraction (PF) derived from P. granatum sarcotesta which is rich in lectin. In addition, the acute oral toxicity, genotoxicity and antigenotoxicity of this protein fraction (PF) from P. granatum sarcotesta was measured. The phytochemical profile of PF was determined using HPLC. The in vitro antioxidant effect was assessed using the methods of total antioxidant capacity (TAC) and DPPH and ABTS+ radical scavenging. Acute oral toxicity was determined in female Swiss mice administered a single dose of 2000 mg/kg. This PF was examined for genotoxicity and antigenotoxicity at doses of 500, 1000 and 2000 mg/kg, utilizing mouse peripheral blood cells. Phytochemical characterization detected a high content of ellagic acid and antioxidant capacity similar to that of ascorbic acid (positive control). PF was not toxic (LD50 >2000 mg/kg) and did not exert a genotoxic effect in mice. PF protected the DNA of peripheral blood cells against damage induced by cyclophosphamide. In conclusion, this PF fraction exhibited significant antioxidant activity without initiating toxic or genotoxic responses in mice.

Carbon ion irradiation combined with PD-1 inhibitor trigger abscopal effect in Lewis lung cancer via a threshold dose.

Background and goal: Carbon ion beam is radio-biologically more efficient than photons and is beneficial for treating radio-resistant tumors. Several animal experiments with tumor-bearing suggest that carbon ion beam irradiation in combination with immunotherapy yields better results, especially in controlling distant metastases. This implies that carbon ion induces a different anti-tumor immune response than photon beam. More complex molecular mechanisms need to be uncovered. This in vivo and in vitro experiment was carried out in order to examine the radio-immune effects and the mechanism of action of carbon ion beam versus X-ray in combination with PD-1 inhibitors. Methods and Materials: Lewis lung adenocarcinoma cells and C57BL/6 mice were used to create a tumor-bearing mouse model, with the non-irradiated tumor growing on the right hind leg and the irradiated tumor on the left rear. 10Gy carbon ion beam or X-ray radiation, either alone or in combination with PD-1 inhibitor, were used to treat the left back tumor. The expression of molecules linked to immunogenicity and the infiltration of CD8+ T lymphocytes into tumor tissues were both identified using immunohistochemistry. IFN-β in mouse serum was measured using an ELISA, while CD8+ T cells in mouse peripheral blood were measured using flow cytometry. Lewis cells were exposed to different dose of X-ray and carbon ion. TREX1, PD-L1, and IFN-β alterations in mRNA and protein levels were identified using Western blot or RT-PCR, respectively. TREX1 knockdown was created by siRNA transfection and exposed to various radiations. Using the CCK8 test, EdU assay, and flow cytometry, changes in cell viability, proliferation, and apoptosis rate were discovered. Results: Bilateral tumors were significantly inhibited by the use of carbon ion or X-ray in combination with PD-1, particularly to non-irradiated tumor(p<0.05). The percentage of infiltrating CD8+ T cells and the level of IFN-β expression were both raised by 10Gy carbon ion irradiation in the irradiated side tumor, although PD-L1 and TREX1 expression levels were also elevated. Lewis cell in vitro experiment further demonstrated that both X-ray and carbon ion irradiation can up-regulate the expression levels of PD-L1 and TREX1 with dose-dependent in tumors, particularly the trend of up-regulation TREX1 is more apparent at a higher dose in carbon ion, i.e. 8 or 10Gy, while the level of IFN-β is decreased. IFN-β levels were considerably raised under hypofractionated doses of carbon ion radiation by gene silencing TREX1. Conclusions: By enhancing tumor immunogenicity and increasing CD8+T infiltration in TME through a threshold dosage, X-ray or carbon ion radiation and PD-1 inhibitors improve anti-tumor activity and cause abscopal effect in Lewis lung adenocarcinoma-bearing mice. TREX1 is a possible therapeutic target and prognostic marker.

Intercellular genetic tracing of cardiac endothelium in the developing heart

Cardiac resident macrophages play vital roles in heart development, homeostasis, repair, and regeneration. Recent studies documented the hematopoietic potential of cardiac endothelium that supports the generation of cardiac macrophages and peripheral blood cells in mice. However, the conclusion was not strongly supported by previous genetic tracing studies, given the non-specific nature of conventional Cre-loxP tracing tools. Here, we develop an intercellular genetic labeling system that can permanently trace heart-specific endothelial cells based on cell-cell interaction in mice. Results from cell-cell contact-mediated genetic fate mapping demonstrate that cardiac endothelial cells do not exhibit hemogenic potential and do not contribute to cardiac macrophages or other circulating blood cells. This Matters Arising paper is in response to Shigeta etal. (2019), published in Developmental Cell. See also the response by Liu and Nakano (2023), published in this issue.

TRAF7 inhibits glycolysis to potentiate growth inhibition and apoptosis of myeloid leukemia cells via regulating the KLF2-PFKFB3 axis

Tumor necrosis factor receptor-related factor 7 (TRAF7) can regulate cell differentiation and apoptosis, but its specific functional mechanism in the pathological process of acute myeloid leukemia (AML) closely related to differentiation and apoptosis disorders is largely unclear. In this study, TRAF7 was found to be lowly expressed in AML patients and a variety of myeloid leukemia cells. TRAF7 was overexpressed in AML Molm-13 and chronic myeloid leukemia (CML) K562 cells by transfection with pcDNA3.1-TRAF7. CCK-8 assay and flow cytometry analysis showed that TRAF7 overexpression induced growth inhibition and apoptosis in K562 and Molm-13 cells. Measurements of glucose and lactate suggested that TRAF7 overexpression impaired glycolysis of K562 and Molm-13 cells. Cell cycle analysis indicated that most of K562 and Molm-13 cells were captured in G0/G1 phase by TRAF7 overexpression. PCR and western blot assay revealed that TRAF7 increased Kruppel-like factor 2 (KLF2) expression but decreased 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression in AML cells. KLF2 knockdown can counteract TRAF7-triggered PFKFB3 inhibition, and abolish TRAF7-mediated glycolysis inhibition and cell cycle arrest. KLF2 knockdown or PFKFB3 overexpression both can partially neutralize TRAF7-induced growth inhibition and apoptosis of K562 and Molm-13 cells. Moreover, Lv-TRAF7 decreased human CD45+ cells in mouse peripheral blood in the xenograft mice established by NOD/SCID mice. Taken together, TRAF7 exerts anti-leukemia effects by impairing glycolysis and cell cycle progression of myeloid leukemia cells via modulating the KLF2-PFKFB3 axis.

Selinexor Prolongs Survival in Patient-Derived Xenograft Models of Down Syndrome Myeloid Leukemia

Background: Selinexor, an inhibitor of the nuclear exportin protein XPO1, has been shown to promote cell cycle arrest and induce apoptosis in acute myeloid leukemia (AML) cells. By inhibiting protein export from the nucleus to the cytoplasm, Selinexor causes nuclear accumulation of proteins including tumor suppressors and translation initiation factor eIF4E, resulting in reduced translation of anti-apoptotic proteins BCL2 and MCL1. Venetoclax, a BCL2 inhibitor, has been considered to synergize with Selinexor in preclinical studies of multiple myeloma (Nguyen et a., Blood, 138:2270, 2021), lymphoma and AML (Fischer et al., Blood Adv, 4:586, 2020). A clinical trial evaluating the combination of Selinexor and venetoclax in relapsed/refractory pediatric AML is currently underway (NCT04898894). However, children with Down syndrome, who have a 500-fold increased risk of developing acute leukemia are excluded from this trial. Methods: The viability of mouse passaged Down syndrome myeloid leukemia (DS-ML) samples following exposure to Selinexor and/or venetoclax was evaluated ex vivo by flow cytometry using annexin V and propidium iodide staining. BLISS synergy scores were calculated using SynergyFinder. For the PDX models, 2-3 million cells were injected intravenously in NSG-SGM3 mice. The percentage of human cells in mouse peripheral blood was determined at periodic intervals by flow cytometry. Mice were randomly recruited to treatment groups (n=5 each) when the percentage of human CD45+ cells were first detected in blood. Selinexor (10 mg/Kg) and venetoclax (100 mg/Kg) were administered by oral gavage for 2 weeks and 4 weeks respectively. Mice were carefully monitored and euthanized at the first instance of pre-determined experimental endpoints. Kaplan-Meier survival estimates were generated using GraphPad Prism. Results: We have generated and extensively characterized patient-derived xenograft models of DS-ML, and used for preclinical evaluation of epigenetic drug combination (azacitidine and panobinostat) (Barwe et al., Blood, 134:2683, 2019), and a combination of azacitidine and venetoclax (Barwe et al., Blood, 138:3452, 2021). Ex vivo studies showed a dose-dependent decrease in the viability of mouse passaged DS-ML cells treated with Selinexor or venetoclax. The estimated EC50 concentrations for Selinexor were 0.31 uM (NTPL-60), 0.53 uM (NTPL-386), and 1.01 uM (15384-029). Venetoclax reduced cell viability at EC50 concentrations ranging from 0.37 uM to 1.54 uM. A combination of these drugs was either additive (NTPL-60), mildly synergistic (NTPL-386) or antagonistic (15384-029) (Figure 1A). We tested the efficacy of Selinexor and venetoclax alone or in combination in vivo in three distinct DS-ML PDX models. Selinexor treatment significantly increased median survival in all three models, while venetoclax was effective in two of three models (Figure 1B, *P<0.05, **P<0.01). However, unlike prior reports in multiple myeloma and AML, addition of venetoclax to the treatment regimen did not provide any survival advantage compared to Selinexor alone. This effect was also observed in the percentage of human CD45+ in blood over time, where human CD45+ blood counts were significantly higher in the combination than Selinexor treatment alone, indicating that Selinexor monotherapy was much more efficient in reducing leukemia burden. Conclusion: We evaluated a combination of Selinexor and venetoclax in DS-ML PDX models ex vivo and in vivo. The combination did not show significant synergy in any of the models ex vivo. Selinexor showed significant improvement in median survival in each of the models and performed better than venetoclax alone. Selinexor venetoclax combination was not beneficial than Selinexor alone in DS-ML PDX models. Studies are in progress to identify the determinants of combinatorial response to Selinexor and venetoclax in DS-ML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Septilin: A versatile anticlastogenic, antigenotoxic, antioxidant and histoprotective herbo-mineral formulation on cisplatin-induced toxicity in mice

Septilin (Spt), a herbo-mineral formulation contains the extracts of Maharasanadi qoath, Tinospora cordifolia, Rubia cordifolia, Emblica officinalis, Moringa pterigosperma, Glycyrrhiza glabra and powders of Balsamodendron mukul and Shankha bhasma. In the present study, the anticlastogenic, antigenotoxic, antioxidant and histoprotective effects of Spt against cisplatin (Csp) induced toxicity in Swiss albino mice were investigated. The micronucleus (MN) test was used to assess the anticlastogenic potential of Spt (125, 250 and 500 mg/kg bw; p.o., 5 days) on somatic cells of mice. The sperm shape abnormality assay detects germinal nuclear damage, which induces spermatogenic dysfunction. Comet assay was employed to study the antigenotoxic potential of Spt on Csp (10 mg/kg bw; i.p.) induced DNA strand breaks in bone marrow cells of mice. The antioxidant enzyme activity of liver superoxide dismutase (SOD) and a biological antioxidant, reduced glutathione (GSH) were measured to determine its hepatoprotective property. The ability of Spt to protect against the histopathologic alterations accompanying Csp-induced liver and testicular injury was studied. The frequencies of MN induced by Csp in bone marrow and peripheral blood cells of mice were significantly decreased by the pre-treatment of Spt. Csp treatment increased the percentage of DNA strand breaks and depleted levels of SOD and GSH content along with histopathological changes. Supplementation of Spt attenuated the toxicity of Csp in liver and testes tissues possible viaimprovement of enzymatic and histological parameters toward normal. This study suggests that the protection offered by Spt against Csp-induced toxicity is partly related to the maintenance of the antioxidant system. Overall, this study shows the protective role of Spt against Csp-induced toxicity in somatic and male germinal cells of mice.

Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML.We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints.Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P<0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. [Display omitted] DisclosuresGopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

Palbociclib and Ponatinib Suppress Acute Myeloid Leukemia in Patient-Derived Xenograft

Aims: Palbociclib, a breast cancer approved CDK4/6 inhibitor, and ponatinib, a BCR/ABL1 inhibitor with a multi-kinase activity approved for chronic myeloid and acute lymphoid leukemia, were previously shown to be effective in vitro against acute myeloid leukemia (AML). Here, we aimed to test this effect in a patient-derived xenograft model.Methods: Two newly diagnosed AMLs (AML #1: myelomonocytic AML, intermediate cytogenetic risk; AML #2: AML with myelodysplasia-related changes, poor cytogenetic risk) were xenotransplanted into NOD SCID gamma mice. Treatment was initiated at detection of approximately 5-20% hCD45+ cells in mouse peripheral blood (PB). Palbociclib, ponatinib, vehicle, and venetoclax as a comparative treatment, were administered orally for 3 weeks, 5 days per week. Chemotherapy (cytarabine+doxorubicine, AraC/Dox) 5+3 regimen served as a positive control. Azacitidine served as another comparative drug and was administered subcutaneously, five days per week in 3 cycles - 1 week on, 1 week off.Results: Significant reduction of disease burden and prolongation of overall survival (OS) were seen with palbociclib and the reference treatment venetoclax in both AMLs, and with chemotherapy in AML#1 (Fig. 1). Ponatinib prolonged OS in AML#1 but failed to provide reduction of disease burden in PB. Interestingly, azacitidine induced the longest remission (<1% hCD45+ cells in PB), for almost 10 weeks, but only in 2/4 mice for AML#2. Treatment toxicity manifested by weight decrease was only seen with chemotherapy (AML#1: 24% weight loss, p = 0.0001; AML#2: 19% loss, p = 0.04), and was also accompanied with early mouse mortality in 3/4 mice for AML#2. None of the tested treatments lead to complete AML eradication and a gradual relapse was seen in PB. The AML infiltration was higher in bone marrow than in spleen at final analysis at relapse (AML#1, 83 vs 73% of hCD45+ cells, mean, p = 0.02; AML#2, 95 vs 65%, p = 0.0001). No changes in AML phenotype were observed between treated and vehicle mice in case of AML#1, except for azacitidine which decreased monocyte (SSCdim, CD45high; 94 vs 96%, mean, p = 0.03), CD34+ (2 vs 5%, p = 0.01) and primitive CD34+CD38- cell (0.1 vs 0.3%, p = 0.005) percentages. For AML#2, blast (SSClow, CD45dim) percentage compared to vehicle (70%) was decreased by chemotherapy (24%, p = 0.01) and increased by venetoclax (80%, p = 0.03) and azacitidine (87%, p = 0.03); monocytes (6% for vehicle) were decreased by chemotherapy (1%, p = 0.004), venetoclax (3%, p = 0.01), and azacitidine (0%, p = 0.008); CD34+ cells were increased by venetoclax (59 vs 39%, p = 0.02).Summary: Palbociclib, and partially ponatinib, demonstrated AML suppression in vivo. This encourages further investigation of their efficacy in different AML subtypes and in combination with other drugs.Funding: Supported by MUNI/A/1595/2020. [Display omitted] DisclosuresMayer: AOP Orphan Pharmaceuticals: Research Funding.

Adenosine monophosphate deaminase 3 null mutation causes reduction of naive T cells in mouse peripheral blood

AbstractAdenosine monophosphate deaminase 3 (Ampd3) encodes the erythrocyte isoform of the adenosine monophosphate (AMP) deaminase gene family. Mutations in this gene have been reported in humans, leading to autosomal-recessive erythrocyte AMP deaminase deficiency. However, the mutation is considered clinically asymptomatic. Using N-ethyl-N-nitrosourea mutagenesis to find mutations that affect peripheral lymphocyte populations, we identified 5 Ampd3 mutations (Ampd3guangdong, Ampd3carson, Ampd3penasco, Ampd3taos, and Ampd3commanche) that strongly correlated with a reduction in naive CD4+ T and naive CD8+ T-cell populations. Causation was confirmed by targeted ablation of Ampd3. Knockout mice had reduced frequencies of CD62LhiCD44lo CD4+ naive and CD8+ naive T cells. Interestingly, these phenotypes were restricted to T cells circulating in peripheral blood and were not seen in T cells from secondary lymphoid organs (lymph nodes and spleen). We found that reduction of naive T cells in the peripheral blood of Ampd3−/− mice was caused by T-cell–extrinsic factor(s), which we hypothesize to be elevated levels of adenosine triphosphate released by Ampd3-deficient erythrocytes. These findings provide an example in which disruption of an erythrocyte-specific protein can affect the physiological status of lymphocytes in peripheral blood.

PDGFRα+ Cell-Derived SCF Regulates Hematopoiesis of Adult Mice

To investigate the effect of PDGFRα+ stromal cells derived SCF on hematopoiesis of adult mice. Pdgfrα-CreER; R26-tdTomato mice model was constructed, and the proportion and distribution of PDGFRα+ cells in the liver, spleen, lung, kidney and bone marrow were analyzed by flow cytometry and confocal microscopy. Then the Pdgfrα-CreER; Scf flox/flox mice model was further constructed, the Scf in PDGFRα+ was knocked out specifically, the effect of Scf-knocked out in PDGFRα+ stromal cells in the propitiation of HSPCs in the bone marrow was analyzed by flow cytometry. The effect of SCF on the proportion on number of peripheral blood cells in mice was analyzed by whole blood analyzer. After Scf was knocked out in PDGFRα+ stromal cells, the propitiation and number of LKS- cell, LKS+ cell, HSC, MPP1, MKP, PreGM, PreMegE, and CFU-E in the bone marrow of mice was decreased, as well as in the number of red blood cells and hemoglobin concentration of peripheral blood. However, Scf knocked out from PDGFRα+ cells showed no effect on the hematopoiesis in spleen. specific knocked out of Scf in PDGFRα+ stromal cells in adult mice can decrease the proportion of HSPCs in the bone marrow and the number of red blood cells in peripheral blood, and finally lead to anemia in mice.

In Vivo Evaluation of Mesothelin As a Therapeutic Target in Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) continues to have a cure rate of only 50% despite the use of highly intensive cytotoxic chemotherapy. Transcriptome sequencing of several AML samples by the NCI/COG TARGET AML Initiative identified mesothelin (MSLN) to be highly overexpressed in about one-third of pediatric AML (Tarlock et al., Blood, 128:2873, 2016). Because MSLN is not expressed in normal bone marrow samples (Fan et al., Blood, 130:3792, 2017) and only to a low level in other human organs and tissues, MSLN is an attractive therapeutic target for pediatric AML (Kaeding et al., Blood, 130:2641, 2017). The anti-MSLN antibody-drug conjugate (ADC) anetumab ravtansine (BAY 94-9343) generated by conjugating MSLN-antibody with tubulin inhibitor DM4 (Meso-ADC), and isotype control antibody conjugated with the same drug (Iso-ADC) were used to evaluate the efficacy of MSLN targeting in vivo. MSLN-overexpressing K562 (K562-MSLN) CML cells and MV4;11 (MV4;11-MSLN) AML cells were generated by lentiviral transduction of MSLN cDNA. Cell line-derived xenografts (CDX) were created by injecting the MSLN-transduced or parental (MSLN-) cells into NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when the median percentage of human cells in mouse peripheral blood was greater than 0.5%. K562-MSLN CDX mice treated with Meso-ADC (5 mg/Kg Q3dx3, i.v.) survived a median of 46 days longer than those treated with Iso-ADC (P=0.0011) and significantly longer than comparison groups, including K562-MSLN CDX mice treated with daunorubicin and Ara-C (DA, P=0.0008) or untreated (P=0.0018) (Fig. 1A). Median survival of K562 CDX mice treated with Meso-ADC, Iso-ADC, or untreated was similar (Fig. 1B). MV4;11-MSLN CDX mice treated with Meso-ADC exhibited complete remission and remained disease-free at 1 year post cell injection, with AML cell burden remaining &amp;lt;0.1% throughout the study period (Fig. 1C). In contrast, MV;11-MSLN CDX mice treated with Iso-ADC or untreated succumbed to disease at 72 and 38 days, respectively. Taken together, these results indicate that Meso-ADC was efficacious in reducing leukemia burden, and this effect required MSLN expression in target cells. We have generated a panel of patient-derived xenograft (PDX) lines by transplanting and serially propagating primary pediatric AML samples into NSG-SGM3 mice. The efficacy of Meso-ADC was also evaluated in a systemic PDX model using a MSLN+ PDX line (NTPL-146). NTPL-146 PDX mice treated with Meso-ADC (5 mg/Kg, Q3dx3 -x2 cycles) survived a median of 50 days longer than those treated with Iso-ADC (P=0.0018, Fig. 1D, arrows indicate time when each treatment cycle was initiated). In an independent experiment with NTPL-146 PDX mice, a survival benefit of Meso-ADC treatment over no treatment was observed after 1 cycle of Meso-ADC treatment (5 mg/Kg, Q3dx3, P=0.0019, Fig. 1E). Additionally, a combination therapy strategy with daunorubicin and Ara-C followed by Meso-ADC (DA -&amp;gt; Meso-ADC) resulted in improved median survival over Meso-ADC (P=0.0027) or DA treatment alone (P=0.0018) (Fig. 1E). The disseminated MSLN+ leukemia mouse models described herein support MSLN-targeted antibody-drug conjugate as a potential treatment strategy in MSLN+ AML. Furthermore, we provide the first in vivo demonstration of synergy between MSLN-targeted therapy and conventional chemotherapy in MSLN+ AML, warranting additional investigation to validate and optimize novel strategies for combination therapy. Figure 1 Disclosures Kaeding: Celgene: Employment. Schatz:Bayer AG: Employment. Sommer:Bayer AG: Employment, Equity Ownership.

Mechanism of CD226-dependant NK cell regulation in mouse obesity

Objective To investigate the effects of CD226 knockout (KO) on obese mice fed with high fat diet and to analyze the composition of immune cells in CD226KO obese mice for further elucidating the immunological mechanism of CD226 involved in high fat diet-induced obesity. Methods Both wild-type (WT) and CD226KO mice were randomly divided into two groups, high-fat and normal diet groups, and fed for 14 weeks to establish the type 2 diabetes model. Immune cells in mouse spleen and peripheral blood were analyzed by flow cytometry. In in vitro experiments, NK92-MI cells were infected with pshRNA-CD226 lentivirus to silence CD226 expression, and then qPCR was performed to detect the expression of Foxp3, TNF-α and IFN-γ at mRNA level. Results In the high-fat diet groups, CD226KO mice had lower blood glucose, serum insulin and HOMA-IR than WT mice, but higher HOMA-IS and HOMA-β. CD226KO could reduce compensatory hyperplasia of islet tissue, and significantly down-regulate the proportion of spleen NK cells in mice. The proportion of CD3- CD49b+ CD25+ Foxp3+ regulatory NK cells (NKreg) increased significantly in CD226KO mice. CD226KO could significantly increase Foxp3 expression in NK92-MI cells and decrease the expression of TNF-α and IFN-γ. Conclusions CD226KO can alleviate insulin resistance, increase the number of islet β-cell and improve islet β-cell function in obese mice. The mechanism might be related to the up-regulation of Foxp3+ NKreg ratio. Key words: CD226; Obesity; Natural killer cell; Regulatory NK cell

Transplantation of skin mesenchymal stem cells attenuated AngII-induced hypertension and vascular injury

Skin mesenchymal stem cells (S-MSCs) revealed an important immunomodulatory activity to markedly suppress the formation of the atherosclerosis (AS) plaque by modulating macrophages, and also inhibit the development of experimental autoimmune encephalomyelitis (EAE) by regulating T helper 17 (Th17) cell differentiation. Macrophages and Th17 cells play important roles in hypertension. However, it remains unclear whether S-MSCs are capable of improving angiotensin (AngII)-induced hypertension by acting on inflammatory cells. Therefore, we studied a direct effect of S-MSC treatment on an AngII-induced hypertensive mouse model. Twenty-seven C57BL/6 (WT) mice were divided into three groups: Control group (WT-NC), AngII-infused group (WT-AngII), and S-MSC treatment group (WT-AngII + S-MSCs). In contrast to WT-AngII group, systolic blood pressure (SBP) and vascular damage were strikingly attenuated after tail-vein injection of S-MSCs. Numbers of Th17 cells in mouse peripheral blood of S-MSC treated group were significantly decreased, and IL-17 mRNA and protein levels were also reduced in the aorta and serum compared with WT-AngII group. Furthermore, macrophages in S-MSC treated group were switched to a regulatory profile characterized by a low ability to produce pro-inflammatory cytokine TNF-α and a high ability to produce anti-inflammatory cytokines Arg1 and IL-10. Mechanistically, we found that S-MSCs inhibited Th17 cell differentiation and induced M2 polarization. Moreover, we found proliferation and migration of S-MSCs were elevated, and expression of CXCR4, the receptor for Stromal derivated factor −1(SDF-1), was markedly increased in lipopolysaccharide (LPS)- stimulated S-MSCs. Given that SDF-1 expression was increased in the serum and aorta in AngII- induced hypertensive mice, the immunomodulatory effects exerted by S-MSCs involved the CXCR4/SDF-1 signaling. Collectively, our data demonstrated that S-MSCs attenuated AngII-induced hypertension by inhibiting Th17 cell differentiation and by modulating macrophage M2 polarization, suggesting that S-MSCs potentially have a role in stem cell based therapy for hypertension.

Water extract of pilose antler can inhibit breast cancer progression of the mouse through modulating its immune system

ABSTRACT Pilose antler (PA) is used to treat many diseases, but its effect on breast cancer is still unclear. Here, we report the effects of PA on the growth of the mouse mammary tumour in vitro and in vivo, and its effects on immune system of the tumor-bearing mice. The 4T1 mouse mammary tumour cells were cultured with media supplemented with water extract of PA (WEPA), and the female BALB/c mice transplanted with 4T1 cells were treated by gavage with WEPA. The results showed that WEPA promoted the proliferation of 4T1 cells in vitro, while inhibited the growth of 4T1 tumour in vivo. In addition, WEPA increased the proportion of CD4+ T cells, CD8+ T cells and NK cells and reduced myeloid-derived suppressive cells in mouse peripheral blood. These results suggest that WEPA can inhibit the growth of mouse 4T1 tumours through modulating immune system of the mouse.

A Combined In Vivo HSC Transduction/Selection Approach Results in Efficient and Stable Gene Expression in Peripheral Blood Cells in Mice

We recently reported on an in vivo hematopoietic stem cell (HSC) gene therapy approach. It involves the subcutaneous injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating helper-dependent adenovirus vector system. HSCs transduced in the periphery homed back to the bone marrow, where they persisted long-term. However, high transgene marking rates found in primitive bone marrow HSCs were not reflected in peripheral blood cells. Here, we tested small-molecule drugs to achieve selective mobilization and transduction of HSCs. We found more efficient GFP marking in bone marrow HSCs but no increased marking in the peripheral blood cells. We then used an in vivo HSC chemo-selection based on a mutant of the O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU and should give stably transduced HSCs a proliferation stimulus and allow for the selective survival and expansion of progeny cells. Short-term exposure of G-CSF/AMD3100-mobilized, in vivo-transduced mice to relatively low selection drug doses resulted in stable GFP expression in up to 80% of peripheral blood cells. Overall, the further improvement of our in vivo HSC transduction approach creates the basis for a simpler HSC gene therapy.

Abstract 1952: Epigenetic modifiers outperform chemotherapy in prolonging survival in patient-derived xenograft models of Down syndrome AML

Abstract Children with Down syndrome (DS) are at a 500-fold increased risk for developing acute myeloid leukemia (AML) before they reach five years of age. DS-AML blasts have somatic mutations in the gene encoding the essential hematopoietic transcription factor GATA-1 resulting in hypersensitivity to chemotherapeutic drugs such as cytarabine and daunorubicin. However, therapy-induced toxicity results in greater morbidity and remains a major barrier in attaining higher survival rate. Thus, alternate therapy approaches to minimize toxicity and increase efficacy are needed. Trisomy 21 and GATA-1 mutations in DS-AML are known to alter the epigenetic landscape in multiple ways. Therefore, we evaluated the efficacy of epigenetic drugs in comparison to chemotherapy in two patient-derived xenograft (PDX) models of DS-AML. We developed two distinct PDX models by successfully engraftment and serial passage of primary DS-AML cells in NSG-B2m mice. Both PDX lines possessed GATA-1 mutation resulting in the expression of a truncated form of GATA-1. NTPL-60 had a nonsense mutation generating a premature stop codon after the initiation codon, while NTPL-386 had a 136 bp deletion in exon 2 resulting in the loss of the initiation codon. The mouse passaged cells were intravenously injected into 6-8 week old NSG-B2m mice. Once disease establishment was confirmed based on the presence of human cells in mouse peripheral blood, five mice per group were treated with vehicle or DNA methylation inhibitor azacitidine and histone deacetylase inhibitor panobinostat either singularly or in combination at a previously determined maximally tolerated dose of 2.5 mg/Kg each. NTPL-386 xenografted mice treated with azacitidine or panobinostat survived 29 and 21 days longer than the vehicle-treated mice respectively, while the mice treated with the combination survived the longest (35 days). Similarly, azacitidine and panobinostat extended the survival of mice transplanted with NTPL-60 by 48 and 31 days respectively compared to the vehicle-treated mice. NTPL-60 mice treated with the epigenetic drug combination are alive at 57 days. Thus, we observed that the azacitidine-panobinostat combination showed statistically significant (p &amp;lt; 0.0001) differences in leukemic burden and mouse survival compared to treatments with either drug alone in both NTPL-386 and NTPL-60 PDX models. We also tested the efficacy of epigenetic therapy followed by chemotherapy. The inclusion of epigenetic therapy before chemotherapy prolonged survival by 39 days compared to vehicle-treated mice. Taken together, our data indicate that epigenetic therapy may be of benefit for the treatment of children with DS-AML. Citation Format: Sonali P. Barwe, E A. Kolb, Anilkumar Gopalakrishnapillai. Epigenetic modifiers outperform chemotherapy in prolonging survival in patient-derived xenograft models of Down syndrome AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1952. doi:10.1158/1538-7445.AM2017-1952

English

Considering the vast use of sweeteners by the Brazilian population and the constant need for toxicological studies of food additives, this study aimed to evaluate the cytotoxic and mutagenic potentials of the sodium saccharin and/or sodium cyclamate sweeteners in plant (Allium cepa) and animal (Mus musculus) test systems based on concentrations permitted by the Brazilian laws. In A. cepa, both the sweeteners individually and their combinations concentration and exposure time (ET: 48, 72 and 168 h) dependently exerted cytotoxic and mutagenic effects. Similarly, an increased in micronuclei formation was also observed by the sweeteners in peripheral blood cells in mice. More toxic effects were observed with the combination doses at 168 ET. There may be a synergistic effect and DNA damage with an increasing concentration and ET. In conclusion, the concentrations considered safe by the Brazilian laws had significant cytotoxic and mutagenic activity on the eukaryotic cells. Key words: Allium cepa, Mus musculus, sweeteners, toxicity.

Mutagenicity assessment of propyl propane thiosulfinate: An organosulfur compound derived from Allium sp.

Davilla nitida and Davilla elliptica (Dilleniaceae) are plants that occur predominantly in the cerrado region of South America. They are used in popular medicine to treat stomach diseases, diarrhea and swelling, particularly of the lymph nodes and testicles. Chemical investigation of these two plant species led to the identification of the compounds myricetin-3-O-α-l-rhamnoside (myricitrin), quercetin-3-O-α-l-rhamnoside (quercitrin), myricetin, quercetin and gallic acid derivatives in the leaves of D. nitida and D. elliptica. Therefore, it was concluded that the two species of Davilla possess qualitatively similar chemical profiles. In the present study, the mutagenic and genotoxic potential of these plants and of their isolated compounds was tested in the Salmonella typhimurium assay (Ames test) with strains TA100, TA98, TA102 and TA97a, in the micronucleus test with peripheral blood cells of mice treated in vivo, and in plasmid DNA to analyze DNA strand-breaks. In the assessment of mutagenic potential by the Ames test, extracts from both plant species and a D. nitida ethyl-acetate fraction induced positive responses. On the other hand, none of the extracts showed genotoxic activity in the mouse cells. In the presence of metal ion, D. nitida and D. elliptica aqueous and ethyl-acetate fractions, as well as their isolated compounds, induced single- and double-strand-breaks in plasmid DNA in a cell-free system.

Effects of immunological and hematological parameter in mice exposed to mixture of volatile organic compounds

Exposure to some kinds of volatile organic compounds (VOCs) leads to immune system disorders, liver and kidney damage, hematological change. However, there is little information about the effect of VOCs mixture on immune system and hematological parameter. In this study, 50 Kunming male mice were exposed in five similar chambers, 0 (control) and four different doses of VOCs mixture (G1–4) for consecutively 10 days at 2 h/day. The concentrations of VOCs mixture were as follows: formaldehyde, benzene, toluene and xylene 1.0 + 1.1 + 2.0 + 2.0, 3.0 + 3.3 + 6.0 + 6.0, 5.0 + 5.5 + 10.0 + 10.0 and 10.0 + 11.0 + 20.0 + 20.0 mg/m3, respectively, which corresponded to 10, 30, 50 and 100 times of indoor air quality standard in china. One day following VOCs exposure, spleen T lymphocyte subpopulation, serum biochemical markers and peripheral blood cells in mice were analyzed, respectively. VOCs exposure decreased significantly erythrocyte count (RBC), platelet (PLT) in peripheral blood in mice. While aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), alkaline phosphatase (ALP) and creatinine (CREA) in serum increased significantly in G4 mice versus controls. Flow cytometry analysis showed that the number of splenic lymphocyte subpopulation cells decreased significantly in G2, 3 and 4 mice in comparison with normal Kunming mice. These results indicate inhalation of VOCs mixture affects CD4/8 subpopulations, liver, kidney function and some hematological parameters in mice.

Aryl Hydrocarbon Receptor Deficiency in an Exon 3 Deletion Mouse Model Promotes Hematopoietic Stem Cell Proliferation and Impacts Endosteal Niche Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor belonging to the Per-Arnt-Sim (PAS) family of proteins. The AHR is involved in hematopoietic stem cell (HSC) functions including self-renewal, proliferation, quiescence, and differentiation. We hypothesize that AHR impacts HSC functions by influencing genes that have roles in HSC maintenance and function and that this may occur through regulation of bone marrow (BM) niche cells. We examined BM and niche cells harvested from 8-week-old AHR null-allele (KO) mice in which exon 3 was deleted in the Ahr gene and compared these data to cells from B6 control mice; young and old (10 months) animals were also compared. We report changes in HSCs and peripheral blood cells in mice lacking AHR. Serial transplantation assays revealed a significant increase in long term HSCs. There was a significant increase in mesenchymal stem cells constituting the endosteal BM niche. Gene expression analyses of HSCs revealed an increase in expression of genes involved in proliferation and maintenance of quiescence. Our studies infer that loss of AHR results in increased proliferation and self-renewal of long term HSCs, in part, by influencing the microenvironment in the niche regulating the balance between quiescence and proliferation in HSCs.