Epigenetic Regulators as the Gatekeepers of Hematopoiesis.

Hematopoiesis is the process by which both fetal and adult organisms derive the full repertoire of blood cells from a single multipotent progenitor cell type, the hematopoietic stem cells (HSCs). Correct enactment of this process relies on a synergistic interplay between genetically encoded differentiation programs and a host of cell-intrinsic and cell-extrinsic factors. These include the influence of the HSC niche microenvironment, action of specific transcription factors, and alterations in intracellular metabolic state. The consolidation of these inputs with the genetically encoded program into a coherent differentiation program for each lineage is thought to rely on epigenetic modifiers. Recent work has delineated the precise contributions of different classes of epigenetic modifiers to HSC self-renewal as well as lineage specification and differentiation into various cell types. Here, we bring together what is currently known about chromatin status and the development of cells in the hematopoietic system under normal and abnormal conditions.

BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity.

Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma.

Craters on the melanoma surface facilitate tumor-immune interactions and demonstrate pathologic response to checkpoint blockade in humans.

Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8 + T cells in the tumor. In zebrafish, CD8 + T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8 + T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response.

Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality.

Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that Calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

End-to-side versus end-to-end neurorrhaphy at the peroneal nerve in rats.

PURPOSE: To evaluate three different kinds of neurorrhaphy of the peroneal nerve. METHODS: Eigthy rats were divided into 5 groups. Control: nerve had no intervention. End-to-end (EE): nerve was cut and elongated with a nerve graft with two end-to-end neurorrhaphies. End-to-side (ES): nerve was cut and sutured to the graft with at the lateral side of the nerve. Side-to-end (SE): the nerve was cut and sutured to the graft with end-to-end neurorrhaphy. Denervated: nerve was cut and both endings were buried into the muscle. The evaluation was done by walking track analysis, electrophysiology, body mass, cranial tibial muscle mass, nerve and muscle fibers morphometry. RESULTS: The EE, ES and SE have the same potential of reinnervation. CONCLUSION: There is no functional or histological difference between these different types of neurorrhaphy.

Low Concentration of 5-Fluorouracil Increases the Effectiveness of Tumor RNA to Activate Murine Dendritic Cells.

AIM: Considering the central role of dendritic cells (DCs) on the development of an antitumor immune response, in this study we used a murine model to evaluate how DC transfection with drug-treated tumor cell RNA changes their phenotype, and whether transfection enhances the in vivo effectiveness of a DC-based antitumor vaccine. MATERIALS AND METHODS: MC-38 colorectal tumor cells were pretreated with the minimum effective concentration of 5-fluorouracil (5-FU), then their total RNA was extracted and transfected into DCs. These DCs were inoculated into C57Bl/6 mice bearing subcutaneous MC-38 tumor. RESULTS: DC transfection with drug-treated tumor RNA increases the percentages of CD40+ (from 37.6% to 61.4%), CD86+ (from 39.8% to 53.4%), and major histocompatibility complex class II+ (from 51.2% to 75.3%) cells, whereas significantly increases the in vivo generation of interferon-γ producer lymphocytes. CONCLUSION: These results reinforce our view that treatment of tumor cells with 5-FU induces transcriptional changes that can be transferred to DCs by RNA transfection, enhancing their ability to stimulate an antitumor response.

Tolerogenic IDO(+) Dendritic Cells Are Induced by PD-1-Expressing Mast Cells.

Mast cells (MCs) are tissue resident cells, rich in inflammatory mediators, involved in allergic reactions, and with an increasingly recognized role in immunomodulation. Dendritic cells (DCs), on the other hand, are central to the determination of immune response patterns, being highly efficient antigen-presenting cells that respond promptly to changes in their microenvironment. Here, we show that direct cell contact between immature monocyte-derived DCs (iDCs) and MC bends DCs toward tolerance induction. DCs that had direct contact with MC (MC-iDC) decreased HLA-DR but increased PD-L1 expression and stimulated regulatory T lymphocytes, which expresses FoxP3(+), secrete TGF-ß and IL-10, and suppress the proliferation of mitogen-stimulated naïve T lymphocytes. Furthermore, MC-iDC expressed higher levels of indoleamine-2,3-deoxigenase (IDO), a phenomenon that was blocked by treatment of MC with anti-PD-1 or by the treatment of DCs with anti-PD-L1 or anti-PD-L2, but not by blocking of H1 and H2 histamine receptors on DCs. Contact with MC also increased phosphorylated STAT-3 levels in iDCs. When a STAT-3 inhibitor, JSI-124, was added to the DCs before contact with MC, the MC-iDC recovered their ability to induce allogeneic T cell proliferation and did not increase their IDO expression.

Synergistic anti-tumor effects of the combination of a benzofuroxan derivate and sorafenib on NCI-H460 human large cell lung carcinoma cells.

Lung cancer is the most frequent and lethal human cancer in the world. Because is still an unsolved health issue, new compounds or therapeutic strategies are urgently needed. Furoxans are presented as potentials candidates for lung cancer treatment. Accordingly, we evaluated the efficacy of a benzofuroxan derivative, BFD-22, alone and combined with sorafenib against NCI-H460 cell line. We showed that BFD-22 has cytotoxic effects on the NCI-H460 cells. Importantly, the Combination Index (CI) evaluation revels that BFD-22 combined with sorafenib has a stronger cytotoxic effect. In addition, the combination induces apoptosis through extrinsic pathway, leading to TRAIL-R1/DR4-triggered apoptosis. Furthermore, BFD-22 combined with sorafenib increases ROS production and simultaneously reduces perlecan expression in the NCI-H460 cells. In accordance, tumor cells were arrested in the S-phase, and these anti-proliferative effects also inhibit cell migration. This is the first study reporting an advantage of BFD-22 combined with sorafenib as a new therapeutic strategy in the fight against lung cancer.

End-to-side versus end-to-end neurorrhaphy at the peroneal nerve in rats

Abstract Purpose: To evaluate three different kinds of neurorrhaphy of the peroneal nerve. Methods: Eigthy rats were divided into 5 groups. Control: nerve had no intervention. End-to-end (EE): nerve was cut and elongated with a nerve graft with two end-to-end neurorrhaphies. End-to-side (ES): nerve was cut and sutured to the graft with at the lateral side of the nerve. Side-to-end (SE): the nerve was cut and sutured to the graft with end-to-end neurorrhaphy. Denervated: nerve was cut and both endings were buried into the muscle. The evaluation was done by walking track analysis, electrophysiology, body mass, cranial tibial muscle mass, nerve and muscle fibers morphometry. Results: The EE, ES and SE have the same potential of reinnervation. Conclusion: There is no functional or histological difference between these different types of neurorrhaphy.