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Inhibition of T-cell proliferation is the most common approach to assess human myeloid-derived suppressor cell (MDSC) functions. However, diverse methodologies hinder the comparison of results obtained in different laboratories. In this chapter, we present a T-cell proliferation assay procedure based on allogeneic MDSC and T-cells that is potentially suitable to multi-center studies. click here The T-cells are isolated from non-cancerous donors and frozen for later use in different research groups. We observed that pure thawed T-cells showed poor proliferative capacities. To retain proliferation, T-cell-autologous mature dendritic cells are supplemented after thawing. MDSC are isolated from clinical samples and represent the sole variant between assays. Flow cytometry is used to assess T-cell proliferation by the dilution of a tracking dye. © 2020 Elsevier Inc. All rights reserved.Immunogenic cell death (ICD), a functionally peculiar type of apoptosis, represents a unique way to deliver danger-associated molecular patterns (DAMPs) to the tumor microenvironment. Once emitted by dying cancer cells, DAMPs orchestrate antigen-specific immune responses by acting on both innate and adaptive components of the immune system. Accumulating preclinical and clinical evidence indicates that one of these DAMPs, calreticulin (CALR) represents a novel powerful prognostic biomarker, reflecting the activation of a clinically relevant anticancer immune response in different cancer malignancies. Therefore, the assessment of CALR emission can provide a therapeutic tool for the stratification of cancer patients and the identification of individuals that are intrinsically capable to respond to a particular treatment. Here we describe methods for the quantification of CALR exposure in the tumor microenvironment of cancer patients by flow cytometry and immunohistochemistry. © 2020 Elsevier Inc. All rights reserved.The progression of cancer is strongly influenced by the crosstalk between cancer cells and immune cells. Immune cells can have both pro- and anti-tumor functions depending on the signals present in the environment. A significant proportion of the immune compartment of most solid tumors consists of tumor-associated macrophages. Although their abundance has been associated with poor prognosis in many solid tumor types, the molecular mechanisms by which cancer cells influence macrophage phenotype and function are largely unknown. In this chapter, we provide a detailed description of in vitro assays to study the impact of cancer cells on macrophages. We provide protocols to obtain macrophages from murine bone marrow and human peripheral blood, and to expose these macrophages to cancer cell-derived secreted molecules using conditioned medium from cancer cells. We describe several assays to assess cancer cell-induced polarization of macrophages. This experimental set-up can be utilized to gain molecular insights into how cancer cells influence macrophages. © 2020 Elsevier Inc. All rights reserved.Tumor-associated macrophages (TAMs) are becoming a promising target for cancer immunotherapy. Significant efforts have been made to study the detrimental role of TAMs both in vivo and in vitro. However, it remains challenging to isolate these macrophages to study their function in human cancers and there is the need to seek alternatives to address these limitations. In this review, we will focus on the three most relevant approaches to obtain in vitro fully differentiated macrophages i.e. peripheral blood, immortalized cell lines such as THP-1 or human induced pluripotent stem cells. We will also provide protocols for the polarization of human macrophages to a TAM-like cells in vitro. © 2020 Elsevier Inc. All rights reserved.Tumor cells treated by immunogenic cell death (ICD) inducers emit danger associated molecular patterns (DAMP), including but not limited to calreticulin (CALR), which translocates from the ER lumen to the surface of the cellular membrane where it serves as de novo uptake signal for antigen presenting cells of the immune system. CALR is exposed at an early stage of ICD and dictates tumor antigen transfer and therefore the immunogenicity of cancer cell death. Here, we provide a bi-color flow cytometry protocol for the quantification of ICD-associated CALR cell surface exposure in fixed samples. As compared to the detection of surface exposed CALR by confocal microscopy, the present flow cytometry-based analysis is cost-efficient and does not require sophisticated equipment. Moreover, the staining panel can be extended to a multicolor analysis for the parallel assessment of additional parameters. © 2020 Elsevier Inc. All rights reserved.After a short summary of Arnold Pick’s biography, the history of how Pick’s disease (PiD) was reported is presented, from its clinical symptoms to its molecular characterization. The macroscopic description of frontotemporal atrophy by Pick is recounted followed by a description of the histological lesions observed by Alzheimer and the progressive characterization of the disease. The subsequent diagnosis has since relied on ultrastructural findings as well as immunohistochemical and biochemical techniques. The discovery of the role of the microtubule-associated τ-protein, encoded by chromosome 17, more specifically of the 3R isoform, has led to the inclusion of PiD in the 3R tauopathies. Today, both sporadic and familial PiDs, including the more frequent behavioral form, are considered as frontotemporal dementias. Experimental models have reproduced some of the lesions but the prion-like hypothesis concerning PiD has not, as yet, been proven.
.AIM Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive degeneration of motor neurons. MicroRNAs are 17 – 27 nucleotide long molecules that regulate post-transcriptional mRNA expression. The aim of this study was to investigate the role of microRNAs in the skeletal muscle of ALS patients and correlate these results with the expression of histone deacetylase 4 (HDAC4) protein. MATERIALS AND METHODS We measured the expression levels of muscle-specific microRNAs (miR-1, miR-133a, miR-133b, miR-206), inflammatory micro-RNAs (miR-27a, miR-221, miR-155), and HDAC4 protein content on western blotting in muscle biopsies obtained for diagnostic reasons in 18 ALS patients 8 genetic forms (C9-ALS and SOD1-ALS), 5 sporadic cases (SALS), and 5 ALS cases affected only by upper motor neuron disease (UMN). RESULTS In muscle of patients with genetic forms of ALS, we found a strong upregulation of miR-206, a muscle-specific miRNA involved in neuromuscular junction (NMJ), regeneration and muscle atrophy, and a decreased expression of HDAC4 protein levels, which is involved both in denervation and regulation of miR-206 in ALS pathophysiology.