There are numerous established techniques for isolating hepatic myeloid cells; however, preserving their phenotypic and functional characteristics can be challenging. We present a straightforward and efficient method to isolate hepatic myeloid cells, including Kupffer cells and lymphocyte antigen 6 complex, locus C+ (Ly6C+) monocytes/macrophages. The procedure involves perfusion of the liver with collagenase and purification with immunomagnetic particles. This protocol ensures the isolation of large quantities of purified, viable, and functional cells without influencing their physiological characteristics. For complete details on the use and execution of this protocol, please refer to Wu et al. (2019).1.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Venetoclax, a BCL-2 inhibitor, has proven to be effective in several hematological malignancies, including mantle cell lymphoma (MCL). However, development of venetoclax resistance is inevitable and understanding its underlying molecular mechanisms can optimize treatment response. We performed a thorough genetic, epigenetic and transcriptomic analysis of venetoclax-sensitive and resistant MCL cell lines, also evaluating the role of the stromal microenvironment using human and murine co-cultures. In our model, venetoclax resistance was associated with abrogated TP53 activity through an acquired mutation and transcriptional downregulation leading to a diminished apoptotic response. Venetoclax-resistant cells also exhibited an upregulation of the PI3K/Akt pathway, and pharmacological inhibition of Akt and ERK with TIC-10 led to cell death in all venetoclax-resistant cell lines. Overall, we highlight the importance of targeted therapies, such as TIC-10, against venetoclax resistance-related pathways, which might represent future therapeutic prospects.

Several environmental stimuli may influence lupus, particularly viral infections. In this study, we used an imiquimod-induced lupus mouse model focused on the TLR7 pathway and proteomics analysis to determine the specific pathway related to viral infection and the related protein expressions in splenic B cells to obtain insight into B-cell responses to viral infection in the lupus model.
We treated FVB/N wild-type mice with imiquimod for 8 weeks to induce lupus symptoms and signs, retrieved splenocytes, selected B cells, and conducted the proteomic analysis. The B cells were co-cultured with CD40L+ feeder cells for another week before performing Western blot analysis. Panther pathway analysis was used to disclose the pathways activated and the protein-protein interactome was analyzed by the STRING database in this lupus murine model.
The lupus model was well established and well demonstrated with serology evidence and pathology proof of lupus-mimicking organ damage. Proteomics data of splenic B cells revealed that the most important activated pathways (fold enrichment > 100) demonstrated positive regulation of the MDA5 signaling pathway, negative regulation of IP-10 production, negative regulation of chemokine (C-X-C motif) ligand 2 production, and positive regulation of the RIG-I signaling pathway. A unique protein-protein interactome containing 10 genes was discovered, within which ISG15, IFIH1, IFIT1, DDX60, and DHX58 were demonstrated to be downstream effectors of MDA5 signaling. Finally, we found B-cell intracellular cytosolic proteins via Western blot experiment and continued to observe MDA5-related pathway activation.
In this experiment, we confirmed that the B cells in the lupus murine model focusing on the TLR7 pathway were activated through the MDA5 signaling pathway, an important RNA sensor implicated in the detection of viral infections and autoimmunity. The MDA5 agonist/antagonist RNAs and the detailed molecular interactions within B cells are worthy of further investigation for lupus therapy.

Diabetes is known to impair the number and function of endothelial progenitor cells in the circulation, causing structural and functional alterations in the micro- and macro-vasculature. The aim of this study was to identify early diabetes-related changes in the expression of genes that have been reported to be closely involved in endothelial progenitor cell migration and function.
Based on review of current literature, this study examined the expression level of 35 genes that are known to be involved in endothelial progenitor cell migration and function in magnetically sorted Lin-/VEGF-R2+ endothelial progenitor cells obtained from the bone marrow of Akita mice in the early stages of diabetes (18 weeks) using RT-PCR and Western blotting. We used the Shapiro-Wilk and D'Agostino & Pearson Omnibus tests to assess normality. Differences between groups were evaluated by Student's t-test for normally distributed data (including Welch correction in cases of unequal variances) or Mann-Whitney test for not normally distributed data.
We observed a significant increase in the number of Lin-/VEGF-R2+ endothelial progenitor cells within the bone marrow in diabetic mice compared with non-diabetic mice. Two genes, SDF-1 and SELE, were significantly differentially expressed in diabetic Lin-/VEGF-R2+ endothelial progenitor cells and six other genes, CAV1, eNOS, CLDN5, NANOG, OCLN and BDNF, showed very low levels of expression in diabetic Lin-/VEGF-R2+ progenitor cells.
Low SDF-1 expression may contribute to the dysfunctional mobilization of bone marrow Lin-/VEGF-R2+ endothelial progenitor cells, which may contribute to microvascular injury in early diabetes.