A DNA vaccine expressing an optimized secreted FAPα induces enhanced anti-tumor activity by altering the tumor microenvironment in a murine model of breast cancer

Cancer-associated fibroblasts (CAFs), major components of the tumor microenvironment (TME), promote tumor growth and metastasis and inhibit the anti-tumor immune response. We previously constructed a DNA vaccine expressing human FAPα, which is highly expressed by CAFs, to target these cells in the TME, and observed limited anti-tumor effects in the 4T1 breast cancer model. When the treatment time was delayed until tumor nodes formed, the anti-tumor effect of the vaccine completely disappeared. In this study, to improve the safety and efficacy, we constructed a new FAPα-targeted vaccine containing only the extracellular domain of human FAPα with a tissue plasminogen activator signal sequence for enhanced antigen secretion and immunogenicity. The number of CAFs was more effectively reduced by CD8⁺ T cells induced by the new vaccine. This resulted in decreases in CCL2 and CXCL12 expression, leading to a significant decrease in the ratio of myeloid-derived suppressor cells in the TME. Moreover, when mice were treated after the establishment of tumors, the vaccine could still delay tumor growth. To facilitate the future application of the vaccine in clinical trials, we further optimized the gene codons and reduced the homology between the vaccine and the original sequence, which may be convenient for evaluating the vaccine distribution in the human body. These results indicated that the new FAPα-targeted vaccine expressing an optimized secreted human FAPα induced enhanced anti-tumor activity by reducing the number of FAPα⁺ CAFs and enhancing the recruitment of effector T cells in the 4T1 tumor model mice.     

CDC25B and CDC25C overexpression in nonmelanoma skin cancer suppresses cell death

Non‐melanoma skin cancer frequently results from chronic exposure to ultraviolet (UV) irradiation. UV‐induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin‐dependent kinase activators, the cell division cycle 25 (CDC25) phosphatases. We previously reported increased CDC25A in nonmelanoma skin cancer, but CDC25B and CDC25C had not been previously examined. Consequently, we hypothesized that increased expression of CDC25B and CDC25C increases tumor cell proliferation and skin tumor growth. We found that CDC25B and CDC25C were increased in mouse and human skin cancers. CDC25B was primarily cytoplasmic in skin and skin tumors and was significantly increased in the squamous cell carcinoma (SCC), while CDC25C was mostly nuclear in the skin, with an increased cytoplasmic signal in the premalignant and malignant tumors. Surprisingly, forced expression of CDC25B or CDC25C in cultured SCC cells did not affect proliferation, but instead suppressed apoptosis, while CDC25C silencing increased apoptosis without impacting proliferation. Targeting CDC25C to the nucleus via mutation of its nuclear export sequence, however, increased proliferation in SCC cells. Overexpression of CDC25C in the nuclear compartment did not hinder the ability of CDC25C to suppress apoptosis, neither did mutation of sites necessary for its interaction with 14‐3‐3 proteins. Analysis of apoptotic signaling pathways revealed that CDC25C increased activating phosphorylation of Akt on Ser⁴⁷³, increased inhibitory phosphorylation of proapoptotic BAD on Ser¹³⁶, and increased the survival protein Survivin. Silencing of CDC25C significantly reduced Survivin levels. Taken together, these data suggest that increased expression of CDC25B or CDC25C are mechanisms by which skin cancers evade apoptotic cell death.     

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 μg, 200 μg and 400 μg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 μg, 200 μg and 400 μg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.     

Left Ventricular Systolic Dysfunction and Cardiovascular Outcomes in Tetralogy of Fallot: Systematic Review and Meta-analysis

BackgroundAlthough there are robust data about the pathophysiology and prognostic implications of left ventricular (LV) systolic dysfunction in patients with acquired heart disease, similar prognostic data about LV systolic dysfunction are sparse in the tetralogy of Fallot (TOF) population. The purpose of this study was to perform a meta-analysis of all studies that assessed the relationship between LV ejection fraction (LVEF) and cardiovascular adverse events (CAEs) defined as death, aborted sudden death, or sustained ventricular tachycardia.MethodsWe used random-effects models to calculate hazard ratios (HRs) and 95% confidence intervals (CIs).ResultsOf the 1,809 citations, 7 studies with 2,854 patients (age 28 ± 4 years) were included. During 5.6 ± 3.4 years' follow-up, there were 82 deaths, 17 aborted sudden cardiac deaths, and 56 sustained ventricular tachycardia events. Overall, CAEs occurred in 5.1% (144 patients). As a continuous variable, LVEF was a predictor of CAE (HR 1.29, 95% CI, 1.09-1.53, P = 0.001) per 5% decrease in LVEF. Similarly, LVEF < 40% was also a predictor of CAE (HR 3.22, 95% CI, 2.16-4.80, P < 0.001).ConclusionsLV systolic dysfunction was an independent predictor of CAE, and we observed a 30% increase in the risk of CAE for every 5% decrease in LVEF, and a 3-fold increase in the risk of CAE in patients with LVEF <40% compared with other patients. These findings underscore the importance of incorporating LV systolic function in clinical risk stratification of patients with TOF and the need to explore new treatment options to address this problem.     

Magnesium lithospermate B acts against dextran sodiumsulfate-induced ulcerative colitis by inhibiting activation of the NRLP3/ASC/Caspase-1 pathway

This study aimed to observe the therapeutic effects of magnesium lithospermate B on acute and chronic colitis induced by dextran sodiumsulfate (DSS) and the role of inflammasome complex (NOD-like receptor protein, NLRP; apoptosis-associated speck-like protein containing, ASC; caspase-1). Establishment of acute and chronic colitis models were by using 5% DSS oral administration in BALB/C male mice. Magnesium lithospermate B (240 mg/kg body weight) was given by subcutaneous injection. Samples were collected for biomarker assay, histological examination, immunohistochemical evaluation and western blot. There was obvious increase in TNF-α level and NLPR3, ASC, and caspase-1 expressions in acute and chronic colitis groups compared with the normal control. Significant decrease of the tumor necrosis factor-α level and the expressions of NLPR3, ASC, and caspase-1 were observed after treatment with magnesium lithospermate B. This study showed that magnesium lithospermate B could be used to treat acute and chronic colitis by inhibiting the activation of the NLRP3/ASC/Caspase-1 pathway.     

IFI 30、PD-L1在人脑胶质瘤中的表达及与病人预后的关系

目的 探讨γ干扰素诱导蛋白30（IFI 30）、程序性死亡因子配体1（PD-L1）在人脑胶质瘤中的表达及与病人预后的关系。方法 选择2015年5月~2019年5月手术切除的103例人脑胶质瘤组织和瘤旁组织，采用免疫组织化学染色检测IFI 30、PD-L1表达情况。随访24个月，记录无进展生存期和总生存期。结果 胶质瘤组织IFI 30、PD-L1高表达率[分别为70.87%（73/103）、68.93%（71/103）]明显高于瘤旁组织[分别为19.42%（20/103）、21.36%（22/103）；P<0.001]。胶质瘤组织IFI 30表达水平与PD-L1表达水平呈明显正相关（r=0.583，P<0.05）。随访24个月，生存75例，死亡28例；多因素logistic回归分析显示，IFI 30高表达、PD-L1高表达是增加病人死亡风险的独立危险因素（P<0.05）。生存曲线分析显示，IFI 30高表达组2年累积生存率（64.52%）和2年累积无进展生存率（65.56%）均明显低于低表达组（分别为82.25%、89.45%；P<0.05）。PD-L1高表达组2年累积生存率（61.78%）和2年累积无进展生存率（60.14%）均明显低于低表达组（分别为88.52%、79.86%；P<0.05）。结论 人脑胶质瘤组织IFI 30、PD-L1呈高表达，与病人不良预后密切相关。     

丹参二萜醌类活性成分通过PKCδ/PKD1μ信号通路抗胰腺癌的作用研究

摘要 目的 验证丹参二萜醌类活性成分对胰腺癌和多发性骨髓瘤的抑制效应，阐明其诱导胰腺癌和多发性骨髓瘤凋亡的作用机制。方法 胰腺癌细胞AsPC-1、BxPC-3用含10% gibco胎牛血清的RPMI1640培养液培养，按隐丹参酮组（30μM）、丹参新酮组（15μM）、去氢丹参新酮组（15μM）分别加药处理。cell-counting-kit-8（CCK8）法检测细胞存活率；AnnexinⅤ-FITC/PI流式细胞术检测细胞凋亡；Western Blot检测相关PKC同工酶磷酸化水平；对AsPC-1和BxPC-3细胞进行siRNA转染，Western Blot检测相关PKC同工酶磷酸化水平。结果 隐丹参酮组AsPC-1、BxPC-3细胞存活率分别为40.1%±5.0%、36.2%±5.4%；丹参新酮组AsPC-1、BxPC-3细胞存活率分别为52.1%±5.1%、47.2%±5.7%；去氢丹参新组AsPC-1、BxPC-3细胞存活率分别为46.1%±5.0%、42.2%±5.4%(P<0.01)。流式细胞术结果显示：AsPC-1组内空白对照组、隐丹参酮组、丹参新酮组、去氢丹参新酮组细胞的凋亡率分别为4.71%、30.10%、52.26%、42.30%；BxPC-3组内空白对照组、隐丹参酮组、丹参新酮组、去氢丹参新酮组细胞的凋亡率分别为5.10%、30.66%、33.76%、51.76%（P<0.01）。Western Blot检测显示隐丹参酮组、丹参新酮组、去氢丹参新酮组较空白对照组，胰腺癌AsPC-1、BxPC-3细胞p-PKD/PKCμ ser916、p-PKCδ thr505、p-PKD/PKCμ ser744/748的水平降低。Western Blot检测显示，siRNA沉默胰腺癌AsPC-1、BxPC-3细胞PKCδ，胰腺癌AsPC-1、BxPC-3细胞PKD/PKCμ ser744/748的磷酸化水平下调。结论 隐丹参酮、丹参新酮、去氢丹参新酮通过抑制PKCδthr505的磷酸化水平，继而PKD1μser744/748磷酸化水平下调，从而显著促进胰腺癌AsPC-1和BxPC-3细胞凋亡发生。     

Combinatorial Inhibition of Myostatin and Activin A Improves Femoral Bone Properties in the G610C Mouse Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a collagen-related bone disorder characterized by fragile osteopenic bone and muscle weakness. We have previously shown that the soluble activin receptor type IIB decoy (sActRIIB) molecule increases muscle mass and improves bone strength in the mild to moderate G610C mouse model of OI. The sActRIIB molecule binds multiple transforming growth factor-β (TGF-β) ligands, including myostatin and activin A. Here, we investigate the musculoskeletal effects of inhibiting activin A alone, myostatin alone, or both myostatin and activin A in wild-type (Wt) and heterozygous G610C (+/G610C) mice using specific monoclonal antibodies. Male and female Wt and +/G610C mice were treated twice weekly with intraperitoneal injections of monoclonal control antibody (Ctrl-Ab, Regn1945), anti-activin A antibody (ActA-Ab, Regn2476), anti-myostatin antibody (Mstn-Ab, Regn647), or both ActA-Ab and Mstn-Ab (Combo, Regn2476, and Regn647) from 5 to 16 weeks of age. Prior to euthanasia, whole body composition, metabolism and muscle force generation assessments were performed. Post euthanasia, hindlimb muscles were evaluated for mass, and femurs were evaluated for changes in microarchitecture and biomechanical strength using micro–computed tomography (μCT) and three-point bend analyses. ActA-Ab treatment minimally impacted the +/G610C musculoskeleton, and was detrimental to bone strength in male +/G610C mice. Mstn-Ab treatment, as previously reported, resulted in substantial increases in hindlimb muscle weights and overall body weights in Wt and male +/G610C mice, but had minimal skeletal impact in +/G610C mice. Conversely, the Combo treatment outperformed ActA-Ab alone or Mstn-Ab alone, consistently increasing hindlimb muscle and body weights regardless of sex or genotype and improving bone microarchitecture and strength in both male and female +/G610C and Wt mice. Combinatorial inhibition of activin A and myostatin more potently increased muscle mass and bone microarchitecture and strength than either antibody alone, recapturing most of the observed benefits of sActRIIB treatment in +/G610C mice. © 2022 American Society for Bone and Mineral Research (ASBMR).