Methods in cell biology最新文献

Alzheimer's disease (AD) is the leading cause of dementia in the elderly, clinically characterized by memory loss, cognitive decline, and behavioral disturbances. Its pathogenesis is not fully comprehended but involves intracellular depositions of amyloid beta peptide (Aβ) and neurofibrillary tangles of hyperphosphorylated tau. Currently, pharmacological interventions solely slow the progression of symptoms. Caenorhabditis elegans (C. elegans) is a simple and valuable organism to study the dynamics of Aβ. It may contribute to advancing our comprehension of AD development and progression, as well as to discovering new treatments. Herein, we describe usual protocols for evaluating Aβ aggregation and toxicity in transgenic C. elegans models of AD (CL2006, CL4176, GMC101, and CL2355 strains) through the visualization and quantification of the peptide with specific fluorescent dyes, in addition to the analysis of particular behaviors (paralysis and chemotaxis associated with learning).

Cancer immunotherapy has been a real revolution and has given many survival chances to several patients. However, the understanding of resistance to immunotherapy is still an unmet need in clinical practice. Monitoring of immune mechanisms could be a tool to better understand this phenomenon. FCM and CyTOF could be used in this field, since they allow the simultaneous analysis of several protein expressions pattern, thus possibly understanding the functions of several immune cell populations, such as T cells, and their interactions with tumor cells and tumor microenvironment. Furthermore, automated cytometry could be used to understand the interaction of drugs with their target through the analysis of receptor occupancy. Spectral overlap, however, could be a limit for multiple simultaneous analyses. Other possible limitations of these techniques are a low number of cells in samples and the need for viable cells (with the possible interference of cell debris). The lack of standardized protocols, and thus the difficult reproducibility, have been the major limit to their application in clinical practice, so international efforts have been made to get to shared guidelines. Ongoing trials are to answer to the possibility of clinical application of these techniques.

9-kDa Granulysin is a protein present in the granules of human activated cytotoxic T lymphocytes and natural killer cells. It has been shown to exert cytolytic activity against a wide variety of microbes: bacteria, fungi, yeast and protozoa. Recombinant isolated granulysin is also capable of inducing tumor cell death, so it could be used as an anti-tumor therapy. Our group has developed granulysin-based immunotoxins in order to target granulysin to tumor cells in vivo. We describe in this chapter the suitable animal model used for testing these immunotoxins against human tumor cells in preclinical assays. This method consists in the xenotransplantation of a given number of human tumor cells subcutaneously in nude mice of the Swiss nu/nu strain or homozygous for the nude gene. Nude mice are immune-deficient, with a very reduced number of T cells, being unable to reject efficiently allo- or xeno-transplanted tissues. Using this approach we follow tumor growth in the different experimental conditions assayed, in the presence or absence of treatment with granulysin alone or with the tumor-directed immunotoxins. We also estimate in this model the possible adverse effects of the treatment in the absence of tumor development. Finally, after sacrifice of the experimentation animals, we use several immunohistochemical assays to study the effect of the treatment on the tumors and the presence of apoptotic cell death markers in the tumor tissue.

B cells exert pulling forces against antigen-presenting cells (APCs) to extract antigens for internalization. The application of tugging forces on B cell receptor (BCR)-antigen bonds promotes discrimination of antigen affinities and sensing of APC physical properties. Here, we describe a protocol for preparing antigen-functionalized DNA tension sensors for quantifying force-mediated antigen extraction in the B cell immune synapse. We describe how to attach the sensors to planar lipid bilayers, quantify their surface density, use them to stimulate B cell activation, and analyze the efficiency of antigen extraction in fixed cells by fluorescence microscopy and image analysis. These techniques should be broadly applicable to studies of force-mediated transfer of molecules in cell-cell contacts.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the principal human pathogens, causing severe infections in skin wounds. MRSA infection triggers a cell response mainly by mitochondrial-mediated pathway, resulting in mitochondrial outer membrane permeabilization, extrusion of the mitochondrial inner membrane into the cytoplasm, and then spillage of mitochondrial DNA (mtDNA) into the cytoplasm. The cell recognizes the discharged cytosolic mtDNA (cmtDNA) as "not-itself" because of mtDNA properties and triggers cascade events, such as the activation of inflammasomes. Here, we detail a method to detect and measure the mtDNA release into the cytoplasm in immortalized keratinocytes (HaCaT cells), after the infection with MRSA at different time points after the infection.

The phenomenon of intercellular transfer of mitochondria has been reported and has attracted significant interest in recent years. The phenomena involve a range of physiological and pathological conditions, such as tumor growth, immunoregulation, and tissue regeneration. There is speculation on the potential restoration of cellular energy status through the transfer of healthy mitochondria from donor cells to cells with impaired mitochondria. Multiple mechanisms and routes of mitochondria transfer have been suggested, including direct cell-to-cell connections, extracellular vesicles, and cell fusion. However, there is limited understanding regarding the precise mechanisms behind mitochondrial transfer, particularly the initiation signals and the associated processes. In order to explore these fundamental mechanisms of mitochondrial transfer, it is imperative to employ techniques that enable direct labeling of mitochondria. Here, we present a detailed methodology utilizing fluorescent protein tagging to visualize mitochondria. The molecular biological techniques applied in this study entail the precise localization of mitochondria with reduced cytotoxicity. This approach facilitates the direct observation of transferred mitochondria through fluorescent and confocal microscopy. The described method can be readily implemented in other mammalian cell types with few modifications, enabling the continuous monitoring of mitochondrial trafficking processes over an extended period.

The externalization of Phosphatidylserine (PS) from the inner surface of the plasma membrane to the outer surface of the plasma membrane is an emblematic event during apoptosis and serves as a potent "eat-me" signal for the efferocytosis of apoptotic cells. Although less well understood, PS is also externalized on live cells in the tumor microenvironment and on live virus-infected cells whereby it serves as an immune modulatory signal that drives tolerance and immune escape. Given the importance of PS in cancer immunology and immune escape, PS-targeting monoclonal antibodies have been characterized with promising immunotherapeutic potential. Here, we describe the cloning and characterization of a series of PS targeting antibodies and their potential use and utility in immuno-oncology.

Combined blockade of the immune checkpoints PD-1 and CTLA-4 has shown remarkable efficacy in patients with melanoma, renal cell carcinoma, non-small-cell lung cancer and mesothelioma, among other tumor types. However, a proportion of patients suffer from serious immune-related adverse events (irAEs). In severe cases, a reduction of the doses or the complete cessation of the treatment is required, limiting the antitumor efficacy of these treatments. Colitis is among the most frequent and problematic irAE associated with immune checkpoint blockade. In this context, animal models that recapitulate the pathophysiological features of immunotherapy-related colitis are needed. In this manuscript, we describe our experience with a mouse model in which the combined CTLA-4 and PD-1 blockade exacerbates the deleterious effects of dextran sulfate sodium (DSS)-induced colitis. This model may constitute a valuable tool for the study of immunotherapy-related colitis.