Mice In Treatment Group Research Articles

Efficiency of resveratrol in the prevention and treatment of age-related hearing loss.

Age-related hearing loss (ARHL) is a major public health concern, which is characterized by gradual, progressive sensorineural hearing loss and deterioration of sound localization, with no effective treatment available to date. The aim of the present study was to evaluate the efficacy of resveratrol to prevent and treat ARHL. For this purpose, 32 male C57BL/6 mice were assigned to four groups: Early treatment, late treatment, control and sham control. The experiment lasted for 15 months. Treatment was started at three months of age in the early treatment group and at sixth months in the late treatment group. The auditory brainstem response test was performed once every three months. At the end of the study period, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, NF-κB, Bcl-2, Bcl-xL, Bax, Bcl-2 homologous antagonist/killer (Bak), caspase-3 and caspase-9 levels in the cochlear tissues of the animals were analyzed by reverse transcription-quantitative PCR. Hearing thresholds of the mice in the early treatment group were better than those in the other groups (P<0.001) at the end of the study. However, hearing levels in the late treatment group were not significantly different from those in the control groups (P>0.05), although mean thresholds were lower. The threshold shift in the early treatment group was significantly lower at all frequencies when compared with those in the control groups (P<0.001). The mRNA expression levels of pro-apoptotic genes Bax and Bak were lower (P<0.05), anti-apoptotic genes Bcl-2 and Bcl-xL were higher (P<0.05), NF-κB, COX-2 and iNOS as genes that have a role in inflammation and caspase-3 and caspase-9 as genes with a vital role in apoptosis were lower (P<0.05) in the early treatment group when compared with the late treatment and control groups. These results suggested that resveratrol is effective in the prevention of ARHL, particularly when started prior to the beginning of hearing loss.

EXTH-80. PBI-200: IN VIVO EFFICACY OF A NOVEL, HIGHLY CNS-PENETRANT NEXT GENERATION TRK INHIBITOR

Abstract TRK kinases (TRK) are clinically validated targets for cancers harboring NTRK gene fusions. However, approved TRK inhibitors (TRKi) give rise to mutations reducing long-term clinical efficacy, and display limited CNS penetration that may impact their ability to address primary brain tumors (PBT), including high-grade gliomas. Second generation compounds in development address some resistance mutations, but are not sufficiently CNS penetrant. PBI-200 is a novel, orally active TRK inhibitor designed to overcome resistance, with high CNS penetration. In vitro assays demonstrate picomolar- to nanomolar potency against both wild-type TRK and the major acquired resistance mutations. A BaF3 xenograft model encoding the LMNA-NTRK1 gene fusion and G595R solvent front mutation showed superior efficacy with PBI-200 relative to first-generation compounds larotrectinib and entrectinib, and equivalent efficacy to the second-generation compound selitrectinib, in terms of tumor growth inhibition. Importantly, drug concentrations in the brains of PBI-200 treated mice showed an approximately 4-fold brain/plasma ratio (BPR). In contrast, BPR of other TRKi were &amp;lt; 0.17, indicating that only PBI-200 achieved sustained drug levels in brain. PBI-200 demonstrated statistically superior CNS efficacy and survival in a KM12-Luc intracranial murine model, as compared to other TRKi (chi-squared 45.6, p &amp;lt; 0.0001). By day 41, 50% of mice in the PBI-200 group remained alive, whereas mice in the other treatment groups had died on or before day 32. Of note, no gross or histopathological CNS adverse effects were observed in a 14d CNS Field Observation Battery study. Finally, PBI-200 was the most selective TRKi in a 125 kinase panel (ThermoFisher) with only 1 off-target kinase inhibited &amp;gt; 60% at 1 micromolar. Together, these data suggest that PBI-200 has potential as a best-in-class, highly CNS-penetrant next generation TRKi. A Phase 1/2 clinical trial evaluating PBI-200 in NTRK fusion-positive patients, including patients with PBT, is ongoing (NCT04901806).

Affibody-Mediated PNA-Based Pretargeted Cotreatment Improves Survival of Trastuzumab-Treated Mice Bearing HER2-Expressing Xenografts.

Treatment of patients with human epidermal growth factor receptor 2 (HER2)-expressing tumors using the monoclonal antibody trastuzumab increases survival. The Affibody-based peptide nucleic acid (PNA)-mediated pretargeted radionuclide therapy has demonstrated efficacy against HER2-expressing xenografts in mice. Structural studies suggest that Affibody molecules and trastuzumab bind to different epitopes on HER2. The aim of this study was to test the hypothesis that a combination of PNA-mediated pretargeted radionuclide therapy and trastuzumab treatment of HER2-expressing xenografts can extend survival compared with monotherapies. Methods: Mutual interference of the primary pretargeting probe ZHER2:342-SR-HP1 and trastuzumab in binding to HER2-expressing cell lines was investigated invitro. Experimental therapy evaluated the survival of mice bearing HER2-expressing SKOV-3 xenografts after treatment with vehicle, trastuzumab only, pretargeting using Affibody-PNA chimera ZHER2:342-SR-HP1 and complementary probe 177Lu-HP2, and combination of trastuzumab and pretargeting. The ethical permit limited the study to 90 d. The animals' weights were monitored during the study. After study termination, samples of liver and kidneys were evaluated by a veterinary pathologist for toxicity signs. Results: The presence of a large molar excess of trastuzumab had no influence on the affinity of ZHER2:342-SR-HP1 binding to HER2-expressing cells invitro. The affinity of trastuzumab was not affected by a large excess of ZHER2:342-SR-HP1 The median survival of mice treated with trastuzumab (75.5 d) was significantly longer than the survival of mice treated with a vehicle (59.5 d). Median survival of mice treated with pretargeting was not reached by day 90. Six mice of 10 in this group survived, and 2 had complete remission. All mice in the combination treatment group survived, and tumors in 7 mice had disappeared at study termination. There was no significant difference between animal weights in the different treatment groups. No significant pathologic alterations were detected in livers and kidneys of treated animals. Conclusion: Treatment of mice bearing HER2-expressing xenografts with the combination of trastuzumab and Affibody-mediated PNA-based radionuclide pretargeting significantly increased survival compared with monotherapies. Cotreatment was not toxic for normal tissues.

Antifatigue effects of Hechong (Tylorrhynchus heterochaetus) through modulation of Nrf2/ARE- mediated antioxidant signaling pathway

This study was to evaluate the antifatigue effect of T. heterochaetus and explore the underlying mechanism of action. T. heterochaetus extract was treated to mice for 28 days. On the 28th day, after weight loaded swimming test. The levels of antioxidant enzymes and levels of pro- and anti-inflammatory cytokines in the liver and muscles of exercised mice were evaluated. mRNA and protein expression levels of Nrf2, SOD, HO-1, and Keap-1 were evaluated using RT-PCR and western blot analysis. The low (2.70 mg/0.5 ml/20 g) and medium (5.41 mg/0.5 ml/20 g) dose enhanced the activities of antioxidant enzymes like SOD, CAT and GPx in the liver and skeletal muscle thereby enhancing the antifatigue effect. The low and medium doses showed good anti-inflammatory effects by evaluating the levels of pro and anti-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-10 both in the liver and skeletal muscle. Furthermore, RT-PCR and western blot analysis showed increased expression of HO-1, SOD, Nrf2, and decreased expression of Keap-1 gene and proteins in liver and skeletal muscle of T. heterochaetus treated group mice. The current results indicate that T. heterochaetus exert the antifatigue effect through attenuating oxidative stress injury and inflammatory responses through the Nrf2/ARE-mediated signaling pathway.

159 METFORMIN DOWNREGULATES PD-L1 EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA BY INHIBITING IL-6 SIGNALING PATHWAY

Abstract ESCC is an aggressive cancer with poor prognosis in China. Metformin with low cost and toxicity have proved to have anti-cancer effects in many kinds of cancers, while its role in combination of immune checkpoint blockade and mechanism in ESCC has seldom been studied. This study was designed to evaluate the potential role of combinatorial anti-tumor effect with metformin and immunomodulatory monoclonal antibodies (mAbs) targeting programmed cell death protein-1 (PD-1) in vitro and in vivo. Methods Reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemistry (IHC) assays were used to study the effects of metformin on the expression levels of PD-L1. In addition, T cell activation and killing assay were performed in the co-culture system of ESCC cell and peripheral blood mononuclear cells (PBMCs) treated with metformin or IL-6 to evaluate the function of inhibiting PD-L1 expression. In vivo assay, we used NPIdKOTM mice model, which was re-constructed replaced immune system by transplanting PBMCs through intravenously injecting. Mice were treated with metformin alone as well as in combination with anti-PD-1 mAbs. Results PD-L1 expression were downregulated by metformin via JAK2 and STAT3 signaling pathway. The results showed that metformin promoted CD3/CD28 induced ERK phosphorylation at a dose dependent measure. And IL-2 was increased in the supernatant of KYSE-450/PBMC coculture system after metformin treatment. Both of which are indicators of T cell activation. T cell mediated tumor killing assay showed that metformin inducing PD-L1 downregulation could protect T cell function, and it could be reversed by IL-6. It showed that metformin enhanced its efficacy in TE-7 cells-harboring mice in the combination treatment group, reflected by tumor sizes assay, and in vivo imaging system. Conclusion Various approaches are under way to expand the benefits and improve the efficacy of these immune checkpoint inhibitors. Another approach is combining immunotherapy with existing anticancer therapies. In this regard, we have evaluated the mechanism of metformin inhibiting PD-L1 expression in ESCC. This finding indicated that metformin significantly improved the antitumor effects by anti-PD-1 blockade without detectable toxicity and suggested that metformin has strong potential to be used in combination with immunotherapy.

Efficacy of low-dose fipronil bait against blacklegged tick (Ixodes scapularis) larvae feeding on white-footed mice (Peromyscus leucopus) under simulated field conditions

BackgroundLyme disease, caused primarily by Borrelia burgdorferi sensu stricto, is the most prevalent vector-borne disease in the United States. Treatment of rodent pathogen reservoirs with an oral acaricide may suppress the production of infected host-seeking ticks posing a risk for human infection. A previous study showed that an oral fipronil bait effectively controlled larval Ixodes scapularis ticks on white-footed mice (Peromyscus leucopus) up to 15 days post-bait exposure. The present study expands upon this finding by exposing group-housed white-footed mice to fipronil bait under simulated field conditions prior to tick infestation.MethodsMice (n = 80) were housed in groups of 10 within large enclosures and offered a choice between fipronil bait within a commercial bait station and an alternative diet. The mice were assigned to two treatment groups and two control groups to undergo bait exposure durations of either 24 h (reduced) or 168 h (extended). Groups were further differentiated by the time point post-bait exposure when larval ticks were applied to mice within feeding capsules (reduced day 1, day 15; extended day 21, day 35). For 4 days post-tick introduction, attached larvae were observed by microscopy and replete larvae were recovered. Replete larvae were monitored for molting success. Plasma was collected from all treatment group mice to obtain fipronil plasma concentrations (CP).ResultsThe fipronil bait (0.005% fipronil) was palatable and controlled larval ticks on white-footed mice when presented under simulated field conditions. Efficacy in preventing attached larvae from feeding to repletion was 100% (day 1), 89.0% (day 15), 85.8% (day 21), and 75.2% (day 35). When also considering molting success, the fipronil bait prevented 100% (day 1), 91.1% (day 15), 91.7% (day 21), and 82.5% (day 35) of larvae attaching to mice from molting. The mean CP per mouse was 191.5 ng/ml (day 1), 29.4 ng/ml (day 15), 10.6 ng/ml (day 21), and 1.0 ng/ml (day 35).ConclusionsThe results suggest that fipronil bait will be consumed by white-footed mice in the presence of an alternative diet, and effectively control larval ticks on treated mice. A field trial is needed to confirm the results of this study. Low-dose fipronil bait may provide a cost-effective means of controlling blacklegged ticks to be integrated into tick management programs.Graphical

Artesunate Restrains Maturation of Dendritic Cells and Ameliorates Heart Transplantation-Induced Acute Rejection in Mice through the PERK/ATF4/CHOP Signaling Pathway.

Background Heart transplantation (HT) is the only effective treatment for end-stage heart failure because it can effectively improve the survival rate and quality of life of patients with heart failure. Artesunate (ART) is an artemisinin derivative, with good water solubility and higher oral bioavailability. The main aim of this study was to determine the role of ART in HT mice. Methods In animal experiments, mice were divided into the control group, HT group, low ART+HT group, and high ART+HT group. Next, inflammatory cell infiltration, oxidative stress injury, and myocardial cell apoptosis were determined in heart tissue. The proportion of multiple lymphocytes in spleen and lymph nodes was then determined using flow cytometry. In addition, cell experiments were conducted to determine the changes in expression of surface maturation markers of BMDC and changes in intracellular reactive oxygen species after LPS stimulation. Finally, western blot analysis was performed to determine the levels of endoplasmic reticulum stress-related proteins (CHOP/ATF4/PERK). Results The survival time of mice in the ART treatment group was significantly prolonged and was positively correlated with the dose. In animal experiments, ART significantly reduced inflammatory cell infiltration in heart tissue and the proportion of CD4+CD8+ T cells in spleens and lymph nodes. Moreover, ART treatment lowered the 8-OHdg in hearts and myocardial apoptosis. In cell experiments, ART treatment slowed down the development and maturation of BMDCs by inhibiting the expression of endoplasmic reticulum stress-related proteins. Furthermore, the treatment alleviated the oxidative stress damage of BMDCs. Conclusion ART can inhibit maturation of dendritic cells through the endoplasmic reticulum stress signaling pathway, thereby alleviating acute rejection in mice after heart transplantation.

Carrageenan oligosaccharides and associated carrageenan-degrading bacteria induce intestinal inflammation in germ-free mice

Carrageenans (CGNs) are widely used in foods and pharmaceuticals although their safety remains controversial. To investigate the effects of CGNs and CGN-degrading bacteria in the human colon, we screened for CGN degradation by human fecal microbiota, and for inflammatory response to CGNs and/or CGN-degrading bacteria in germ free mice. Thin-layer chromatography indicated that high molecular weight (MW) CGNs (≥100 kDa) remained undegraded in the presence of human fecal microbiota, whereas low MW CGNs, i.e., κ-carrageenan oligosaccharides (KCO, ~4.5 kDa) were degraded when exposed to seven of eight human fecal samples, although sulfate groups were not removed during degradation. Bacteroides xylanisolvens and Escherichia coli isolates from fecal samples apparently degraded KCO synergistically, with B. xylanisolvens serving as the primary degrader. Combined treatment of KCO with KCO-degrading bacteria led to greater pro-inflammatory effects in the colon and rectum of germ-free mice than either KCO or bacteria alone. Similarly, p-p38-, CD3-, and CD79a-positive immune cells were more abundant in combined treatment group mice than in either single treatment group. Our study shows that KCO-degrading bacteria and the low MW products of KCO can promote proinflammatory effects in mice, and represent two key markers for evaluating CGN safety in foods or medicines.

Effects of Caper (Capparis Spinosa) and Acetic Acid on Lipid Profile and Protein Concentration in the Serum of Albino Mice

This study aimed to determine the effects of alcoholic and aqueous extracts of caper (Capparis Spinosa) and acetic acid on serum lipid profile and proteins levels in mice. Sixty adult mice with an average weight of 24±4 g grams were divided into four groups (15 mice for each). The first group (G1) was administrated daily with an oral dose of caper alcoholic extract (200 mg/kg) for 28 days. The second group (G2) was administrated daily with an oral dose of caper aqueous extract (200 mg/kg) for 28 days. The third group (G3) was administrated with a daily dose of 10 % acetic acid (2 ml/kg) for 28 days. The fourth Group (G4) was administrated daily with distilled water for 28 days, as a control group. The levels of lipid profile parameters, blood urea, total protein, albumin, and globulin were determined. The results showed a significant reduction (P≤0.05) in cholesterol and triglyceride levels in mice that were treated with alcohol or aqueous extracts of caper compared with acetic acid-treated and control groups. On the other hands, the results showed a significant reduction (P≤0.05) of blood urea levels in mice that were treated with alcohol or aqueous extracts of caper compared with acetic acid-treated and control groups. While the results recorded non-significant differences in the levels of total protein, albumin, and globulin in the serum of mice of different treatment groups. From the results, it can be concluded that caper has protective effects via acting to improve the lipid profile and urea level in the blood of mice.

Invivo Effect Analysis of Rhodomyrtus tomentosa Leaf Ethanol Extract Against Escherichia coli

Puerperium infection contributed 11 percent of 48.17 percent of maternal mortality during the puerperium period; one of the causes of increased risk of puerperium infection was bacteria Gram-negative Escherichia coli. Puerperium infection occurred because bacteria enter through the vagina, wounds on the insertion of the placenta, and other parts spread to blood throughout the body and damage the spleen, liver, and uterus. This research aimed to determine the effect of Rhodomyrtus tomentosa leaves ethanol extract (Rtl-EE) as an antibacterial on the spleen, liver, and uterus of Escherichia coli -induced puerperal infection model mice. Mice in the postnatal treatment group were induced by Escherichia coli bacteria intravaginally and given different doses of ethanol extract of Rhodomyrtus tomentosa leaves. Examination of bacterial colonies growth used pour plate method with EMBA media. The research results showed that the administration of Rtl-EE decreased the growth of bacterial colonies respectively in the liver 1280, 924, 330 CFU/mL; the uterus 1806, 1180, 874 CFU/mL; and the spleen 712, 112, 774 CFU/mL. Colony growth started to decrease at dose 1 of Rtl-EE (100mg/kg BW) of mice model of Escherichia coli -induced puerperal infection. Rhodomyrtus tomentosa leaves contain active compounds - phenols, flavonoids, saponins, tannins, alkaloids, and triterpenoids as antibacterial. Rtl-EE had an antibacterial effect by reducing the number of bacteria on the spleen, liver, and uterus of Escherichia coli -induced puerperal infection model mice. Further research needs to conduct to examine the toxic dose of Rhodomyrtus tomentosa leaves against Escherichia coli bacteria.

Intravenous Administration of Toll-Like Receptor Inhibitory Peptide 1 is Effective for the Treatment of Systemic Lupus Erythematosus in a Mus musculus Model.

Systemic lupus erythematosus (SLE) is a common chronic autoimmune inflammatory disease According to recent studies, signaling through Toll-like receptor (TLR) protein, which promotes the production of inflammatory cytokines, leads to the development of SLE TLR-inhibitory peptide 1 (TIP1) has been newly identified for the treatment of autoimmune diseases. The effect of TIP1 was analyzed in an SLE mouse model (MRL/lpr) The mice in the control treatment group (n=5) were administered an intravenous injection of phosphate-buffered saline twice weekly, whereas the mice in the TIP1 treatment group (n=6) were administered an intravenous injection of TIP1 (1 nmol/g) twice weekly MRL/mpj mice (n=5) were selected as normal controls The mice were injected for 4 weeks between 14 and 18 weeks of age, followed by assays of their spleen, kidneys, lymph nodes, serum, and urine. The antinuclear antibody and inflammatory cytokine (interferon-α) in the serum as well as levels of albumin in the urine of the mice in the TIP1 treatment group had decreased when compared to those of mice in the control treatment group Kidney inflammation in mice in the TIP1 treatment group was alleviated The mRNA expression levels of TLR7- or TLR9-related downstream signaling molecules also decreased in all organs of the mice in the TIP1 treatment group. Intravenous treatment with TIP1 reduces symptoms and markers of inflammation in MRL/lpr mice Hence, TIP1 is a promising medication for the treatment of SLE.

Chemoprotective Effect of Daphnetin in Doxorubicin Treated Esophageal Cancer Stem Cell Xenograft Tumor Mouse.

Chemotherapy drugs commonly used for cancer therapy, but chemotherapy has limitation due to side effects. Current studies suggest natural products are reducing the side effects of chemotherapy medicines. In this study, we examined the side effects of doxorubicin (Dox) in esophageal cancer cells (CSCs) derived tumors in vivo. Esophageal cancer cells (YMI) were treated in vitro with daphnetin (DAP) along with DOX. The MTT assay was used for estimating the cell viability and Annexin/7-AAD was used for the determination of apoptosis. Cell cycle arrest was conducted using the PI-staining method. The potential effect of DAP was evaluated by the estimation of oxidative stress such as total antioxidant capacity (TAC), malondialdehyde (MDA) and superoxide dismutase (SOD) and body weight in the xenograft mice. DAP can protect Dox cell toxicity by suppressing cell apoptosis of ESCC. DAP arrest the cells as S-phase. In vivo experimental study showed that Dox simultaneously with DAP decreases the tumor size along with increased body weight in the nude mice compared to Dox alone treated group mice. Dox along with the DAP exhibited less systemic toxicity and reduced oxidative stress fraction circulation. The result suggests that daphnetin may be used as an adjuvant therapy to reduce the systemic toxicity of chemotherapeutic agents, such as DOX, in stem cell treatment with ESCC cancer.

Evaluation of the in vivo antihypertensive effect and antioxidant activity of HL-7 and HL-10 peptide in mice.

The tendency to use bioactive peptides has increased in recent decades, and research would be essential for recognizing the therapeutic effects of peptides present in animals or food resource. In this study, the in vivo antioxidant and antihypertensive properties of peptides HL-7 with the sequence of YLYELR and HL-10 with the sequence of AFPYYGHHLG were identified from scorpion venom of H. lepturus were evaluated. To study the in vivo effects of peptides, D-galactose-induced and DOCA salt-induced mice models were used. The results of the antioxidant assay for both peptides showed that the activity of serum and liver catalase (CAT), as well as superoxide dismutase (SOD) enzymes, was significantly decreased in the D-galactose-induced group (NC), while MDA levels were increased in serum and the liver tissue samples (p < 0.01). Compared with the D-galactose-induced mice, the peptide treated mice group had a higher activity of antioxidant enzymes namely CAT and SOD, as well as a lower lipid peroxidation level. Also, the results of antihypertensive activity for both peptides showed that systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the mice treated with the HL-7 and HL-10 peptides were significantly reduced in a dose-dependent manner (p < 0.01). The administration of the HL-7 peptide at doses of 2mg/kg BW (LP1), 5mg/kg BW (-IP1) and 15mg/kg BW (HP1) significantly diminished the mean arterial blood pressure (MAP) by 11mmHg, 31mmHg and 40.47mmHg, respectively. Accordingly, treatment of mice with the HL-10 peptide at doses of 2mg/kg BW (LP2), 5mg/kg BW (IP2) and 15mg/kg BW (HP2) considerably lowered the MAP by 8mmHg, 18.3mmHg and 21.93mmHg, respectively. Our findings suggest that both the HL-7 and HL-10 peptides could be potentially utilized as antihypertensive and antioxidant components.

CAR T Cells Targeting FLT3 on AML Confer Potent Anti-Leukemic Activity and Act Synergistically with the FLT3 Inhibitor Midostaurin

Background: FMS-like tyrosine kinase 3 (FLT3) is a homodimeric transmembrane protein that is expressed on normal hematopoietic stem (HSC) and progenitor cells. FLT3 is also uniformly present on malignant blasts in acute myeloid leukemia (AML) providing a target for immunotherapy with chimeric antigen receptor (CAR)-modified T cells. FLT3 is a driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3-ITD) and other gain-of-function mutations in the intracellular tyrosine kinase domain. Accordingly, the FLT3 inhibitor midostaurin has recently been approved in combination with chemotherapy to treat FLT3-mutated AML. Here, we explored the antileukemia efficacy of FLT3 CAR-T cells against FLT3 wild-type (wt) and FLT3-ITD+ AML, alone and in combination with midostaurin to determine whether the two therapeutic modalities could be used synergistically, particularly in high-risk FLT3-ITD+ AML.Methods: A FLT3-CAR (BV10 scFv_IgG4 hinge_CD28_CD3ζ_EGFRt) was encoded in the lentiviral vector epHIV7 for gene-transfer into T cells of healthy donors and AML patients (n>6). CAR-T cell cytolytic activity was evaluated in FACS-/luminescence-based assays, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. Immunodeficient NSG mice were engrafted with MOLM-13/ffLuc AML cells and treated with 5x106 CAR-modified or control T cells (CD4:CD8 ratio = 1:1). MOLM-13 cells were cultured in the presence of 10nM midostaurin and doses titrated up to 50nM to induce resistance (MOLM-13R).Results: We confirmed specific recognition and high-level cytolytic activity of CD8+ FLT3 CAR-T cells against a panel of AML cell lines that included THP-1 (FLT3 wt), MOLM-13 (FLT3-ITD+/-) and MV4;11 (FLT3-ITD+/+), as well as FLT3-ITD+ primary AML blasts obtained from multiple patients. Both CD8+ and CD4+ FLT3 CAR-T cells produced IFN-γ and IL-2, and underwent proliferation after stimulation with native AML cell lines and primary AML blasts.We then treated AML cell lines with midostaurin and detected a significant increase in FLT3 expression in MOLM-13 and MV4;11 by flow cytometry (delta MFI = 8986 and 1442, respectively, p<0.05), but not THP-1 cells, indicating that upregulation specifically occurred in FLT3-ITD+ AML. We sought to determine whether higher FLT3 expression on midostaurin-resistant AML target cells translated into superior antileukemia efficacy of FLT3 CAR-T cells. Indeed, we observed superior cytolytic activity, cytokine production and proliferation of FLT3 CAR-T cells after stimulation with MOLM-13R compared to native MOLM-13 cells (n=3 experiments; p<0.05). In contrast to FLT3, expression of CD33 and CD123 as alternative CAR target antigens was decreased on MOLM-13R.We confirmed upregulation of FLT3 occurred on native MOLM-13 AML cells in vivo during midostaurin treatment in NSG mice, and observed synergistic antileukemia efficacy of FLT3 CAR-T cells and midostaurin in combination therapy. Intriguingly, we detected higher frequencies of CAR-T cells in all mice that received the combination treatment, compared to mice that received FLT3 CAR-T cells alone. At the end of the observation period, all mice in the combination treatment group had achieved complete remission of AML from peripheral blood, bone marrow and spleen.Importantly, we also show that FLT3 CAR-T cells recognize normal HSCs and interfere with hematopoiesis in colony formation assays in vitro . However, midostaurin treatment did not lead to an increase of FLT3 on normal HSC consistent with their expression of wt FLT3.Summary: Collectively, our data demonstrate that FLT3 CAR-T cells mediate potent reactivity against FLT3 wt and FLT3-ITD+ AML cells in vitro and in vivo . We further demonstrate that FLT3 CAR-T cells and midostaurin act synergistically, i.e. midostaurin-induced upregulation of FLT3 in FLT3-ITD+ AML cells enhances their recognition and elimination by FLT3 CAR-T cells. Due to recognition of normal HSC, the clinical use of FLT3 CAR-T cells is likely restricted to a defined therapeutic window prior to allogeneic HSC transplantation, followed by CAR-T cell depletion and hematopoietic reconstitution. Our data provide an operational model to augment the antileukemia efficacy of FLT3 CAR-T cells in high-risk FLT3-ITD+ AML patients, and to mitigate the risk for relapse with FLT3-negative AML variants that may otherwise develop under therapeutic pressure. DisclosuresNo relevant conflicts of interest to declare.

Targeting T-Cell Malignancies with Chimeric Antigen Receptors Using Two Structurally Different Antigen Binding Domains Against CD5

Patients with relapsed T-cell acute lymphoblastic leukemia (T-ALL) have extremely poor survival when treated with chemotherapy alone. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a chance of cure for these patients, but attaining clinical remission in order to undergo HSCT remains the biggest therapeutic challenge. Chimeric antigen receptor (CAR) T-cell therapy has been used with great success in relapsed/refractory B-ALL patients. The same approach, however, is difficult in targeting T-cell disease given the lack of a T-lymphoblast specific surface antigen. The use of natural killer (NK) cells as the effector cell provides a potential solution. We are testing two different CAR structures directed against CD5, a pan T-cell marker; one consisting of a more traditional immunoglobulin (Ig) based single chain variable fragment (scFv), while the other comprising of a variable lymphocyte receptor (VLR) as the antigen recognition domain. VLRs are single chain crescent shaped proteins that represent the functional unit of the adaptive immune system in jawless vertebrates. Our previous work has shown that a VLR can be used as an alternative for CAR-based antigen recognition. Here, we directly compare a VLR to its scFv counterpart in a CAR setting. The single chain structure of a VLR allows for the rapid creation of novel CARs, compared to the corresponding use of a scFv-CAR, in which the variable heavy and light sequences need further engineering for adapting to CAR technologies. Thus, our hypothesis was that natural killer cells engineered to express an anti-CD5 CAR can specifically be used to target T-cell malignancies, with the VLR-CAR being equal or superior to scFv-based cassettes. We constructed two second generation CARs using the VLR or scFv sequence targeting CD5 as our antigen recognition domain. Both CD5-CAR constructs showed similar activity when tested in CD5-positive Jurkat cells. For our cytotoxicity studies, we used the IL-2 dependent natural killer cell line NK-92. To generate uniform populations of CAR-expressing NK cells we transduced NK-92 cells with a dual lentiviral construct expressing the CD5-CAR and green fluorescent protein (GFP), and then selected for the positively transduced cells using flow sorting for GFP. In vitro cytotoxicity studies showed significantly increased killing using the CD5-CAR expressing NK-92 cells compared to unmodified NK-92 cells (p<0.05), for both VLR and scFv modified cells. We then tested the two CD5-CARs in a T-cell leukemia mouse model. NOD scid gamma (NSG) mice were intravenously injected with luciferase expressing Jurkat leukemia cells to establish the T-cell leukemia xenograft model. Four doses of NK-92 cells were injected over a two week period starting on day 7 post-tumor injection. Bioluminescence imaging was used to monitor tumor burden. The CD5-scFv-CAR NK-92 treated mice group had a significant survival advantage over the other groups (p<0.01). Interestingly, the CD5-VLR-CAR NK-92 treated mice did not have a significant advantage over naïve NK-92 or saline treated groups. Given the superior in vivo cytotoxicity demonstrated in our studies, we plan to use the CD5-scFV-CAR going forward as we transition to primary cells. Overall, these studies provide the foundation to move these studies into preclinical testing as a treatment for relapsed T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Protective effects of glycine against lipopolysaccharide-induced intestinal apoptosis and inflammation.

Intestinal dysfunction is commonly observed in humans and animals. Glycine (Gly) is a functional amino acid with anti-inflammatory and anti-apoptotic properties. The objective of this study was to test the protective effects of Gly against lipopolysaccharide (LPS)-induced intestinal injury. 28 C57BL/6 mice with a body weight (BW) of 18 ± 2g were randomly assigned into four groups: CON (control), GLY (orally administered Gly, 5g/kg BW/day for 6days), LPS (5mg/kg BW on day 7, i. p.), and GLY + LPS (Gly pretreatment and LPS administration). Histological alterations, inflammatory responses, epithelial cell apoptosis, and changes of the intestinal microbiota were analyzed. Results showed that, compared with the CON group, mice in the LPS treatment group showed decreased villus height, increased crypt depth, and decreased ratio of villus height to crypt depth, which were significantly attenuated by Gly. Neither LPS nor Gly treatment altered morphology of the distal colon tissues. LPS increased the apoptosis of jejunum and colon epithelial cells and protein abundance of cleaved caspase3 in the jejunum, which were markedly abrogated by Gly. LPS also elevated the mRNA levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MYD88), pro-inflammatory cytokines, and chemokines in the jejunum and colon. These alterations were significantly suppressed by Gly. In addition, Gly supplementation attenuated infiltration of CD4+, CD8+ T-lymphocytes, CD11b+ and F4/80+ macrophages in the colon. Furthermore, Gly increased the relative abundance of Mucispirillum, Lachnospiraceae-NK4A136-group, Anaerotruncus, Faecalibaculum, Ruminococcaceae-UCG-014, and decreased the abundance of Bacteroides at genus level. Supplementation with Gly might be a nutritional strategy to ameliorate LPS-induced intestinal injury in mice.

FC 098MESENCHYMAL STEM CELL EXOSOMES AMELIORATE MICE PERITONEAL FIBROSIS INDUCED BY HUMAN PERITONITIS DIALYSIS EFFLUENT

Abstract Background and Aims Peritoneal fibrosis is a severe complication of peritoneal dialysis, but there are few effective therapies for it. The purpose of this study was to investigate the protective effect of exosomes secreted by mouse bone marrow mesenchymal stem cells on peritoneal fibrosis and to reveal the mechanism. Method Forty-two male C57BL/6 mice were randomly divided into a normal group, a control group (2.5% glucose dialysate), a peritonitis-effluent group (The overnight 2.5% glucose dialysate of patients with peritonitis), a high glucose dialysate group (4.25% glucose concentration), a peritonitis-dialysate + exosome group, and a high glucose dialysate + exosome group. The mouse model of peritoneal fibrosis was constructed by intraperitoneal injection of human peritonitis effusion continuously for 42 days. The mice in the exosome treatment group received intraperitoneal injection of mesenchymal stem cell (MSC)-exosomes twice. The level of peritoneal structural and functional damage, inflammation, fibrosis and mesothelial cell damage of peritoneum were detected. Furthermore, the effect of MSC-exosomes was validated in vitro. Results Peritoneal transport was significantly impaired and peritoneal thickness was significantly increased in the peritonitis-effluent group and the high glucose dialysate group after 42 days. The degree of peritoneal inflammation and fibrosis in the two groups was significantly higher than the control group. The results suggested that human peritonitis dialysis effluent could be used to construct a mouse model of peritoneal fibrosis. Masson staining results showed that the fibrosis degree of peritonitis - effluent + exosome group was significantly less than the peritonitis - effluent group. Immunohistochemical analysis showed that the expression levels of mesothelial markers E-cadherin and ZO-1, neutrophil granulocytes (MPO) and macrophages (F4/80), and fibrosis markers collagen I and a-SMA in the peritonitis - effluent + exosome group were significantly lower than those in the peritonitis - effluent group. Similarly, the high glucose dialysate + exosome group mice showed significantly lower levels of peritoneal inflammation and fibrosis than the high glucose dialysate group mice. In vitro experiments showed that exosomes could down-regulate the secretion of IL-1, IL-6 and TGF- by renal tubular cells stimulated by high glucose dialysate, maintain the expression of mesenchymal cell marker (E-cadherin), and inhibit the mesenchymal marker (-SMA), suggesting that exosomes could inhibit the transdifferentiation of peritoneal mesenchymal cell-mesenchymal cells (MMT). Conclusion MSC-exosomes can alleviate peritoneal fibrosis by inhibiting peritoneal mesothelial cell-mesenchymal cell transdifferentiation.

Dietary supplementation with N-3 polyunsaturated fatty acid-enriched fish oil promotes wound healing after ultraviolet B-induced sunburn in mice.

N‐3 polyunsaturated fatty acids (n‐3 PUFA) can alleviate ultraviolet B (UVB)‐induced skin cancers, but their effects on sunburn and upcoming wound healing remain controversial. This study aimed to explore the impact of n‐3 PUFA‐enriched fish oil (n‐3 PUFA‐FO) on UVB‐induced sunburns and subsequent healing. Sixty C57BL/6 female mice were divided into two groups. The treated group mice were fed n‐3 PUFA‐FO for the entire duration of the experiment. Mice in the control group were fed a standard diet. After two weeks of n‐3 PUFA‐FO feeding, mice were exposed to UVB for 20 min and sacrificed 20 d later. Skin photodamage and lesion area were recorded during wound healing. Epidermal lesion thickness was quantified in hematoxylin and eosin‐stained skin sections. Inflammation and macrophage polarization were assessed by qRT‐PCR. Oxidative stress and antioxidant enzyme activity were quantified using specific ELISA kits. N‐3 PUFA‐FO feeding decreased UVB photodamage and accelerated wound healing progression, both of which were coupled with less intense inflammation and increased macrophage M2 phenotype polarization. Furthermore, n‐3 PUFA‐FO brought about a decrease in malondialdehyde (MDA) levels but increased the activity of catalase (CAT) and glutathione peroxidase (GP), without changing superoxide dismutase (SOD) activity. N‐3 PUFA‐FO protects against UVB‐induced skin photodamage and promotes wound healing by modulating macrophage phenotypic polarization and antioxidant enzyme activities. N‐3 PUFA‐FO could be a novel therapeutic approach for both the prevention and treatment of sunburns.

The Effect and Mechanism of Hyperin on Ovarian Reserve of Tripterygium Glycosides-Induced POI Mice

To investigate the effect and mechanism of hyperin on the improvement of ovarian reserve of tripterygium glycosides (TG)-induced primary ovarian insufficiency (POI) in mice. Adult female BALB/c mice were used as research subjects and were randomly assigned to the control group, POI model group and hyperin treatment group, with 40 mice in each group. TG was given at 40 mg/kg twice a day by gavage for 2 weeks to create the POI mouse model. Mice in the hyperin treatment group were given hyperin at 75 mg/(kg·d) by gavage for 4 weeks after the model was established. The body mass of the mice was weighed and the gonadal index was calculated. Ovarian histological changes were observed by HE staining, and the number of follicles at all levels was calculated. Serum estradiol (E 2), follicle-stimulating hormone (FSH), anti-mullerian hormone (AMH), superoxide dismutase (SOD) and catalase (CAT) were assessed with ELISA. The mRNA and protein levels of nuclear factor (erythroid-derived 2)-related factor 2 (Nrf-2), heme oxygenase-1 (HO-1), Caspase3, Bcl-2 and Bax in ovarian granulosa cells were measured by RT-qPCR and Western blot. The protein levels of phosphorylated phosphatidylinositol 3-hydroxy kinase (p-PI3K) and phosphorylated protein kinase B (p-Akt) were measured by Western blot. The reactive oxygen species (ROS) levels in granulosa cells were determined by H2DCFDA. The apoptosis of granulosa cells was examined by TUNEL assay. Compared with mice in the POI model group, the body mass and gonadal index of hyperin-treated mice increased (all P<0.05). The pathological damage of the ovary decreased, and the number of follicles at all levels and corpora lutea increased (all P<0.05). Serum E 2, AMH, SOD and CAT levels increased, and FSH level decreased (all P<0.05). At the molecular level, the expression of Nrf-2, HO-1, p-PI3K, p-Akt and Bcl-2 in ovarian granulosa cells increased, while the expression of Caspase3 and Bax decreased (all P<0.05). ROS level decreased ( P<0.05). TUNEL assay showed reduced apoptosis of granulosa cells ( P<0.05). Hyperin improved ovarian reserve in TG-induced POI mice through Nrf-2/HO-1antioxidant stress response and the anti-apoptotic effect of PI3K/Akt pathways.

Manipulations of the gut microbiome alter chemotherapy-induced inflammation and behavioral side effects in female mice

Chemotherapy treatment is associated with acute behavioral side effects (fatigue, anorexia) that significantly reduce patient quality of life and are dose-limiting, thereby increasing mortality (Kidwell et al., 2014). Disruptions to gut homeostasis (diarrhea, constipation, microbial dysbiosis) are also observed in patients receiving chemotherapy. In non-oncological patients, facets of mental health (fatigue, anxiety, depression) correlate with alterations in the gut microbiome, suggestive of a contribution of the gut in CNS disease etiology. The potential gut-to-brain pathway is poorly understood in patients receiving chemotherapy. Our prior studies have demonstrated a correlation between chemotherapy treatment, gut changes, peripheral and central inflammation, and behavioral symptoms in mice. Here we aimed to determine the extent to which chemotherapy-associated gut manipulations modulate the behavioral and biological consequences of chemotherapy. We measured sickness behaviors, peripheral and central inflammatory mediators, and anxiety in conventional or germ-free female mice: 1) cohabitating with mice of the opposite treatment group, 2) pre-treated with broad-spectrum antibiotics, or 3) given an intra-gastric gavage of gut content from chemotherapy-treated mice. In cohabitation studies, presumed coprophagia promoted body mass recovery, however strong associations with inflammation and behavior were not observed. Reduction of gut microbial alpha diversity via antibiotics did not prevent chemotherapy-associated side effects, however the relative abundances of the genera Tyzzerella, Romboutsia, and Turicibacter correlated with circulating inflammatory (IL-1β) and behavioral outcomes (lethargy, anxiety-like behavior). A gut microbiota transplant from chemotherapy-treated mice decreased central locomotion in open field testing, increased circulating CXCL1, and increased hippocampal Il6 and Tnfa in germ-free mice compared to germ-free mice that received a transplant from vehicle-treated mice. Taken together, these data provide further evidence that the gut microbiota likely contributes to the development of chemotherapy-associated side effects. This work has significant implications in the future treatment of anxiety in patients, and warrants future studies using microbe-based treatment options.