Mechanical Properties Affect Primary T Cell Activation in 3D Bioprinted Hydrogels.

T cells play a critical role in the adaptive immune response of the body, especially against intracellular pathogens and cancer. In vitro, T cell activation studies typically employ planar (two-dimensional, 2D) culture systems that do not mimic native cell-to-extracellular matrix (ECM) interactions, which influence activation. The goal of this work was to study T cell responses in a cell line (EL4) and primary mouse T cells in three-dimensional (3D) bioprinted matrices of varied stiffness. Cell-laden hydrogels were 3D bioprinted from gelatin methacryloyl (GelMA) using a digital light processing (DLP)-based 3D bioprinter operated with visible light (405 nm). Mechanical characterization revealed that the hydrogels had pathophysiologically relevant stiffnesses for a lymph node-mimetic tissue construct. EL4, a mouse T cell lymphoma line, or primary mouse T cells were 3D bioprinted and activated using a combination of 10 ng/mL of phorbol myristate acetate (PMA) and 0.1 µM of ionomycin. Cellular responses revealed differences between 2D and 3D cultures and that the biomechanical properties of the 3D bioprinted hydrogel influence T cell activation. Cellular responses of the 2D and 3D cultures in a soft matrix (19.83 ± 2.36 kPa) were comparable; however, they differed in a stiff matrix (52.95 ± 1.36 kPa). The fraction of viable EL4 cells was 1.3-fold higher in the soft matrix than in the stiff matrix. Furthermore, primary mouse T cells activated with PMA and ionomycin showed 1.35-fold higher viable cells in the soft matrix than in the stiff matrix. T cells bioprinted in a soft matrix and a stiff matrix released 7.4-fold and 5.9-fold higher amounts of interleukin-2 (IL-2) than 2D cultured cells, respectively. Overall, the study demonstrates the changes in the response of T cells in 3D bioprinted scaffolds toward engineering an ex vivo lymphoid tissue-mimetic system that can faithfully recapitulate T cell activation and unravel pathophysiological characteristics of T cells in infectious biology, autoimmunity, and cancers.

High throughput screening identifies auranofin and pentamidine as potent compounds that lower IFN-γ-induced Nitric Oxide and inflammatory responses in mice: DSS-induced colitis and Salmonella Typhimurium-induced sepsis.

Interferon-gamma (IFN-γ) is a type II interferon produced primarily by T cells and natural killer cells. IFN-γ induces the expression of inducible nitric oxide synthase (NOS2) to catalyze Nitric Oxide (NO) production in various immune and non-immune cells. Excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including peritonitis and inflammatory bowel diseases. In this study, we screened the LOPAC®1280 library in vitro on the H6 mouse hepatoma cell line to identify novel non-steroidal small molecule inhibitors of IFN-γ-induced NO production. Compounds with the highest inhibitory activity were validated, which led to identifying the lead compounds: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC50 and goodness of fit analyses. Mechanistic investigations revealed that majority of the lead compounds suppress the IFN-γ-induced transcription of Nos2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1, Socs1 and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced reactive oxygen species amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in resident as well as thioglycolate-elicited peritoneal macrophages (PMs). Finally, in vivo testing of the lead compounds in the pre-clinical DSS-induced ulcerative colitis mice model revealed pentamidine and auranofin to be the most potent and protective lead compounds. Also, pentamidine and auranofin greatly increase the survival of mice in another inflammatory model: Salmonella Typhimurium-induced sepsis. Overall, this study identifies novel anti-inflammatory compounds targeting IFN-γ-induced NO-dependent processes to alleviate two distinct inflammatory models of disease.

Dynamic changes in thymic sub-populations during acute and long-term infections with virulent and virulence-attenuated Salmonella Typhimurium strains in C57BL/6 and autoimmune-prone lpr mice.

SALMONELLA: Typhimurium infection in mice results in drastic loss of immature CD4- CD8- double negative (DN) and CD4+ CD8+ double positive (DP) thymic subsets compared to mature single positive (SP) subsets. We investigated changes in thymocyte sub-populations post infection with a wild type (WT) virulent strain and ΔrpoS, a virulence-attenuated strain, of Salmonella Typhimurium in C57BL/6 (B6) and Fas-deficient autoimmune-prone lpr mice. The WT strain caused acute thymic atrophy with greater loss of thymocytes in lpr mice compared to B6 mice. Infection with ΔrpoS caused progressive thymic atrophy in B6 and lpr mice. Analysis of thymocyte subsets revealed that immature thymocytes including the DN, immature single positive (ISP), and DP thymocytes underwent extensive loss. SP thymocytes were more resistant to loss in WT-infected B6 mice, whereas WT-infected lpr and ΔrpoS-infected mice exhibited depletion of SP thymocytes. Overall, thymocyte sub-populations exhibited differential susceptibilities depending on bacterial virulence and the host background.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice.

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice. This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival. Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Notch Signalling: A Potential Therapeutic Pathway in Oral Squamous Cell Carcinoma.

Cancer, a set of diseases characterized by abnormal cell growth resulting from alteration in the expression pattern of diverse genes, is one of the prominent causes of mortality and morbidity worldwide. This modification of various genes leads to altered signalling cascades and changes in the molecular network. These changes eventually give rise to cellular dysfunction and then to systemic failure causing death. Of the several pathways that are aberrantly activated in cancer, Notch signalling pathway is a prominent one. Notch signalling pathway is a juxtracrine signalling pathway which activates the genes associated with cell proliferation, survival and angiogenesis. Notch signalling pathway components are seen to be upregulated in several types of cancer. Oral squamous cell carcinoma (OSCC) is a predominant category of oral cancer where aberrant activation of Notch signalling causes tumour progression and metastasis. In this review, we discuss the Notch signalling pathway, its components, forms and its role in the progression and metastasis of oral squamous cell carcinoma.

Comprehending the crosstalk between Notch, Wnt and Hedgehog signaling pathways in oral squamous cell carcinoma - clinical implications.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a malignant oral cavity neoplasm that affects many people, especially in developing countries. Despite several advances that have been made in diagnosis and treatment, the morbidity and mortality rates due to OSCC remain high. Accumulating evidence indicates that aberrant activation of cellular signaling pathways, such as the Notch, Wnt and Hedgehog pathways, occurs during the development and metastasis of OSCC. In this review, we have articulated the roles of the Notch, Wnt and Hedgehog signaling pathways in OSCC and their crosstalk during tumor development and progression. We have also examined possible interactions and associations between these pathways and treatment regimens that could be employed to effectively tackle OSCC and/or prevent its recurrence. CONCLUSIONS: Activation of the Notch signaling pathway upregulates the expression of several genes, including c-Myc, ß-catenin, NF-κB and Shh. Associations between the Notch signaling pathway and other pathways have been shown to enhance OSCC tumor aggressiveness. Crosstalk between these pathways supports the maintenance of cancer stem cells (CSCs) and regulates OSCC cell motility. Thus, application of compounds that block these pathways may be a valid strategy to treat OSCC. Such compounds have already been employed in other types of cancer and could be repurposed for OSCC.

Mitochondrial dynamics, positioning and function mediated by cytoskeletal interactions.

The ability of a mitochondrion to undergo fission and fusion, and to be transported and localized within a cell are central not just to proper functioning of mitochondria, but also to that of the cell. The cytoskeletal filaments, namely microtubules, F-actin and intermediate filaments, have emerged as prime movers in these dynamic mitochondrial shape and position transitions. In this review, we explore the complex relationship between the cytoskeleton and the mitochondrion, by delving into: (i) how the cytoskeleton helps shape mitochondria via fission and fusion events, (ii) how the cytoskeleton facilitates the translocation and anchoring of mitochondria with the activity of motor proteins, and (iii) how these changes in form and position of mitochondria translate into functioning of the cell.

Conventional and Emerging Markers in Stem Cell Isolation and Characterization.

Stem cells have emerged as a promising source of cell-based therapy in regenerative medicine with several stem cell-based products currently in clinical trials. Despite the immense therapeutic potential, their isolation from some of the emerging sources and their characterization has been naïve owing to the lack of standard markers for the same. Some biomarkers have now been well established for the isolation and characterization of stem cells. However, there are emerging markers that can be used in addition to these conventional markers or independent of them to establish the identity of the stem cells. In this review, an attempt has been made to describe a few conventionally used markers and emerging markers for the identification, isolation and characterization of stem cells from various niches across the three germ layer origins.

Mitochondrial Superoxide Dismutase Specifies Early Neural Commitment by Modulating Mitochondrial Dynamics.

Studies revealing molecular mechanisms underlying neural specification have majorly focused on the role played by different transcription factors, but less on non-nuclear components. Earlier, we reported mitochondrial superoxide dismutase (SOD2) to be essential for self-renewal and pluripotency of mouse embryonic stem cells (mESCs). In the present study, we found SOD2 to be specifically required for neural lineage, but not the meso- or endoderm specification. Temporally, SOD2 regulated early neural genes, but not the matured genes, by modulating mitochondrial dynamics-specifically by enhancing the mitochondrial fusion protein Mitofusin 2 (MFN2). Bio-complementation strategy further confirmed SOD2 to enhance mitochondrial fusion process independent of its antioxidant activity. Over-expression of SOD2 along with OCT4, but neither alone, transdifferentiated mouse fibroblasts to neural progenitor-like colonies, conclusively proving the neurogenic potential of SOD2. In conclusion, our findings accredit a novel role for SOD2 in early neural lineage specification.

Hypoxia induced EMT: A review on the mechanism of tumor progression and metastasis in OSCC.

Hypoxia, a condition of low oxygen tension in tissues, has emerged as a crucial factor in tumor pathophysiology. Hypoxic microenvironment gives rise to altered cellular metabolism and triggers varied molecular responses. These responses promote tumor progression and confer radiation resistance and chemo resistance to tumors. The predominant molecules that are associated with hypoxia research are the hypoxia inducible factors (HIFs). HIFs are known to regulate a large group of genes that are involved in cell survival, proliferation, motility, metabolism, pH regulation, extracellular matrix function, inflammatory cell recruitment and angiogenesis by inducing the expression of their downstream target genes. The process of epithelial to mesenchymal transition (EMT) has been associated with metastasis in cancer. Reports also suggest that hypoxia triggers EMT in several types of cancer including breast cancer, prostate cancer and oral cancer. Oral cancer is a predominant cancer in Central and South East Asia. However, in the recent times, the incidence rates of oral cancer have been increasing in Northern and Eastern Europe as well. This review articulates the role of hypoxia and the associated factors like HIFs in inducing EMT in oral cancer (OSCC).

Clinical Applications of Induced Pluripotent Stem Cells - Stato Attuale.

In an adult human body, somatic stem cells are present in small amounts in almost all organs with the function of general maintenance and prevention of premature aging. But, these stem cells are not pluripotent and are unable to regenerate large cellular loss caused by infarctions or fractures especially in cells with limited replicative ability such as neurons and cardiomyocytes. These limitations gave rise to the idea of inducing pluripotency to adult somatic cells and thereby restoring their regeneration, replication and plasticity. Though many trials and research were focused on inducing pluripotency, a solid breakthrough was achieved by Yamanaka in 2006. Yamanaka's research identified 4 genes (OCT-4, SOX-2, KLF-4 and c-MYC) as the key requisite for inducing pluripotency in any somatic cells (iPSCs). Our study, reviews the major methods used for inducing pluripotency, differentiation into specific cell types and their application in both cell regeneration and disease modelling. We have also highlighted the current status of iPSCs in clinical applications by analysing the registered clinical trials. We believe that this review will assist the researchers to decide the parameters such as induction method and focus their efforts towards clinical application of iPSCs.

Eminent Sources of Adult Mesenchymal Stem Cells and Their Therapeutic Imminence.

In the recent times, stem cell biology has garnered the attention of the scientific fraternity and the general public alike due to the immense therapeutic potential that it holds in the field of regenerative medicine. A breakthrough in this direction came with the isolation of stem cells from human embryo and their differentiation into cell types of all three germ layers. However, the isolation of mesenchymal stem cells from adult tissues proved to be advantageous over embryonic stem cells due to the ethical and immunological naivety. Mesenchymal Stem Cells (MSCs) isolated from the bone marrow were found to differentiate into multiple cell lineages with the help of appropriate differentiation factors. Furthermore, other sources of stem cells including adipose tissue, dental pulp, and breast milk have been identified. Newer sources of stem cells have been emerging recently and their clinical applications are also being studied. In this review, we examine the eminent sources of Mesenchymal Stem Cells (MSCs), their immunophenotypes, and therapeutic imminence.