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.     

Aloe-Emodin Protects RIN-5F (Pancreatic β-cell) Cell from Glucotoxicity via Regulation of Pro-Inflammatory Cytokine and Downregulation of Bax and Caspase 3

To determine the protective effect of aloe-emodin (AE) from high glucose induced toxicity in RIN-5F (pancreatic β-cell) cell and restoration of its function was analyzed. RIN-5F cells have been cultured in high glucose (25 mM glucose) condition, with and without AE treatment. RIN-5F cells cultured in high glucose decreased cell viability and increased ROS levels after 48 hr compared with standard medium (5.5 mM glucose). Glucotoxicity was confirmed by significantly increased ROS production, increased pro-inflammatory (IFN-γ, IL-1β,) & decreased anti-inflammatory (IL-6&IL-10) cytokine levels, increased DNA fragmentation. In addition, we found increased Bax, caspase 3, Fadd, and Fas and significantly reduced Bcl-2 expression after 48 hr. RIN-5F treated with both high glucose and AE (20 μM) decreased ROS generation and prevent RIN-5F cell from glucotoxicity. In addition, AE treated cells cultured in high glucose were transferred to standard medium, normal responsiveness to glucose was restored within 8hr and normal basal insulin release within 24 hr was achieved when compared to high glucose.     

Central nervous system and peripheral cell labeling by vascular endothelial cadherin-driven lineage tracing in adult mice

Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vascular cell types,we performed cell lineage tracing analysis using transgenic mice engineered to express a fluorescent marker following activation by tamoxifen in vascular endothelial cadherin promoter-expressing cells(VEcad-CreERT2;B6 Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze).Activation of target-cell labeling following 1.5 months of ad libitum feeding with tamoxifen-laden chow in 4–5 month-old mice resulted in the tracing of central nervous system and peripheral cells that include:cerebellar granule neurons,ependymal cells,skeletal myocytes,pancreatic beta cells,pancreatic acinar cells,tubular cells in the renal cortex,duodenal crypt cells,ileal crypt cells,and hair follicle stem cells.As Nestin expression has been reported in a subset of endothelial cells,Nes-CreERT2 mice were also utilized in these conditions.The tracing of cells in adult Nes-CreERT2 mice revealed the labeling of canonical progeny cell types such as hippocampal and olfactory granule neurons as well as ependymal cells.Interestingly,Nestin tracing also labeled skeletal myocytes,ileal crypt cells,and sparsely marked cerebellar granule neurons.Our findings provide support for endothelial cells as active contributors to adult tissue progenitor pools.This information could be of particular significance for the intravenous delivery of therapeutics to downstream endothelial-derived cellular targets.The animal experiments were approved by the Boise State University Institute Animal Care and Use Committee(approval No.006-AC15-018)on October 31,2018.     

Diagnostic challenges for a novel SH2D1A mutation associated with X‐linked lymphoproliferative disease

Mutations in SH2D1A, encoding the intracellular adaptor signaling lymphocyte activation molecule associated protein (SAP), are associated with X‐linked lymphoproliferative disease type 1 (XLP1). We identified a novel hemizygous SH2D1A c.49G > A (p.E17K) variant in a 21‐year‐old patient with fatal Epstein‐Barr virus infection–associated hemophagocytic lymphohistiocytosis. Cellular and biochemical assays revealed normal expression of the SAP variant protein, yet binding to phosphorylated CD244 receptor was reduced by >95%. Three healthy brothers carried the SH2D1A c.49G > A variant. Thus, data suggest that this variant represents a pathogenic mutation, but with variable expressivity. Importantly, our results highlight challenges in the clinical interpretation of SH2D1A variants and caution in using functional flow cytometry assays for the diagnosis of XLP1.     

原肌球蛋白4在胃癌细胞迁移、侵袭和转移中的作用

目的:探索原肌球蛋白4（Tropomyosin4,TPM4）在胃癌细胞的迁移、侵袭和转移中的作用。方法:利用Real-time PCR、Western Blot及免疫荧光，检测原肌球蛋白4在不同胃癌细胞系及正常胃上皮细胞中的表达情况；利用免疫组化，检测原肌球蛋白4在胃癌组织及癌旁正常组织中的表达情况；利用慢病毒技术，在GC9811-P细胞中，分别转染LV-TPM4、shRNA-TPM4及对照空载体；通过Transwell和体内实验，检测胃癌细胞迁移、侵袭和转移。结果:Real-time PCR、Western Blot及免疫组化结果显示，原肌球蛋白4在胃癌细胞系及胃癌组织中的表达降低；上调原肌球蛋白4的表达后，抑制胃癌细胞的迁移、侵袭和转移；下调原肌球蛋白4的表达后，促进胃癌细胞的迁移、侵袭和转移（P<0.05）。结论:原肌球蛋白4抑制胃癌细胞的迁移、侵袭和转移。     

HIFα Regulates Developmental Myelination Independent of Autocrine Wnt Signaling

The developing CNS is exposed to physiological hypoxia, under which hypoxia-inducible factor α (HIFα) is stabilized and plays a crucial role in regulating neural development. The cellular and molecular mechanisms of HIFα in developmental myelination remain incompletely understood. A previous concept proposes that HIFα regulates CNS developmental myelination by activating the autocrine Wnt/β-catenin signaling in oligodendrocyte progenitor cells (OPCs). Here, by analyzing a battery of genetic mice of both sexes, we presented in vivo evidence supporting an alternative understanding of oligodendroglial HIFα-regulated developmental myelination. At the cellular level, we found that HIFα was required for developmental myelination by transiently controlling upstream OPC differentiation but not downstream oligodendrocyte maturation and that HIFα dysregulation in OPCs but not oligodendrocytes disturbed normal developmental myelination. We demonstrated that HIFα played a minor, if any, role in regulating canonical Wnt signaling in the oligodendroglial lineage or in the CNS. At the molecular level, blocking autocrine Wnt signaling did not affect HIFα-regulated OPC differentiation and myelination. We further identified HIFα–Sox9 regulatory axis as an underlying molecular mechanism in HIFα-regulated OPC differentiation. Our findings support a concept shift in our mechanistic understanding of HIFα-regulated CNS myelination from the previous Wnt-dependent view to a Wnt-independent one and unveil a previously unappreciated HIFα–Sox9 pathway in regulating OPC differentiation.SIGNIFICANCE STATEMENT Promoting disturbed developmental myelination is a promising option in treating diffuse white matter injury, previously called periventricular leukomalacia, a major form of brain injury affecting premature infants. In the developing CNS, hypoxia-inducible factor α (HIFα) is a key regulator that adapts neural cells to physiological and pathologic hypoxic cues. The role and mechanism of HIFα in oligodendroglial myelination, which is severely disturbed in preterm infants affected with diffuse white matter injury, is incompletely understood. Our findings presented here represent a concept shift in our mechanistic understanding of HIFα-regulated developmental myelination and suggest the potential of intervening with an oligodendroglial HIFα-mediated signaling pathway to mitigate disturbed myelination in premature white matter injury.     

The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire

Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.     

Suppression of PRDX4 inhibits cell proliferation and invasion of ectopic endometrial stromal cells in endometriosis

Abstract

Oxidative stress (OS) has been proposed to play a role in the development of EMs. Peroxiredoxins are a family of antioxidant proteins that exhibit peroxidase activity in a thioredoxin-dependent manner, protecting cells against OS. The Western blotting results showed that the relative expression of PRDX4 was significantly increased in ectopic endometria compared with the normal endometria of EMs-free (p?<?.05). The H₂O₂ concentration was also significantly higher in the ectopic endometrium. PRDX4 siRNA was transfected into primary ectopic endometrial stromal cells (EESCs). The viability of the transfected EESCs was measured by CCK-8 assay, and the results showed significantly decreased cell viability. Furthermore, the apoptosis rate and ROS generation in flow cytometry assays were significantly increased after the knockdown of PRDX4 expression (p?<?.05). Scratch assays and transwell assays revealed that decreased expression of PRDX4 mediated by siRNA inhibited EESC migration and invasion. In conclusion, these findings indicate the potential role of PRDX4 in the development of EMs and PRDX4 as a possible therapeutic target for EMs treatment.