An In Vitro Device for Evaluation of Cellular Response to Flows Found at the Apex of Arterial Bifurcations

Apoptosis, along with proliferation, is a form of lymphocyte response to activating stimuli. In the early stages of cell differentiation, the apoptotic response prevails and it results to the formation of tolerance to inductor antigen. Mature lymphocytes proliferate in response to stimulation and it means the initial stage in the development of the immune response. Since in this case apoptosis and proliferation acts as alternative processes, their ratio can serve as a measure of the effectiveness of the cellular response to activating signals. The resistance of autoreactive T-cells to apoptosis is the main key point in the development of type 1 diabetes mellitus (T1DM). Autoreactive T-cells migrates from the bloodstream to the islet tissue of the pancreas and take an active part in b cells destruction. The resistance of autoreactive effector T-cells to apoptosis may suggest their high proliferative potential. Therefore, the comparative evaluation of apoptosis and proliferation of peripheral blood lymphocytes can give a more complete picture of their functional state and thus will help to reveal the causes of ineffective peripheral blood T-ceiis apoptosis in patients with T1DM and will help to understand more deeply the pathogenesis of the disease. in this article, the features of proliferative response of peripheral blood T-cells in patients with T1DM and in individuals with high risk of developing T1DM have been studied. Apoptosis of T-cell subpopulations has been investigated. The correlation between the apoptotic markers and the intensity of spontaneous and activation- induced in vitro T-cells proliferation of was revealed. it was determined, that autoreactive peripheral blood T-cells were resistant to apoptosis and demonstrated the increased proliferative potential in patients with T1DM and in individuals with high risk of developing T1DM.

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A rapid, parasite-dependent cellular response to Dirofilaria immitis in the Mongolian jird (Meriones unguiculatus)

Parasites & Vectors ◽

10.1186/s13071-020-04455-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Christopher C. Evans ◽

Katherine M. Day ◽

Yi Chu ◽

Bridget Garner ◽

Kaori Sakamoto ◽

...

Keyword(s):

Cellular Response ◽

Dirofilaria Immitis ◽

Cell Attachment ◽

Early Response ◽

Meriones Unguiculatus ◽

Filarial Parasite ◽

Immune Mediated ◽

Secretory Products ◽

Species Specific

Abstract Background The Mongolian jird (Meriones unguiculatus) has long been recognized as a permissive host for the filarial parasite Brugia malayi; however, it is nonpermissive to another filarial parasite, canine heartworm (Dirofilaria immitis). By elucidating differences in the early response to infection, we sought to identify mechanisms involved in the species-specific clearance of these parasites. We hypothesized that the early clearance of D. immitis in intraperitoneal infection of the jird is immune mediated and parasite species dependent. Methods Jird peritoneal exudate cells (PECs) were isolated and their attachment to parasite larvae assessed in vitro under various conditions: D. immitis and B. malayi cultured separately, co-culture of both parasites, incubation before addition of cells, culture of heat-killed parasites, and culture with PECs isolated from jirds with mature B. malayi infection. The cells attaching to larvae were identified by immunohistochemistry. Results In vitro cell attachment to live D. immitis was high (mean = 99.6%) while much lower for B. malayi (mean = 5.56%). This species-specific attachment was also observed when both filarial species were co-cultured, with no significant change from controls (U(9, 14) = 58.5, p = 0.999). When we replicated these experiments with PECs derived from jirds subcutaneously infected with B. malayi, the results were similar (99.4% and 4.72% of D. immitis and B. malayi, respectively, exhibited cell attachment). Heat-killing the parasites significantly reduced cell attachment to D. immitis (mean = 71.9%; U(11, 14) = 7.5, p < 0.001) while increasing attachment to B. malayi (mean = 16.7%; U(9, 15) = 20, p = 0.002). Cell attachment to both species was reduced when larvae were allowed a 24-h pre-incubation period prior to the addition of cells. The attaching cells were identified as macrophages by immunohistochemistry. Conclusions These results suggest a strongly species-dependent response from which B. malayi could not confer protection by proxy in co-culture. The changes in cell attachment following heat-killing and pre-incubation suggest a role for excretory/secretory products in host immune evasion and/or antigenicity. The nature of this attachment is the subject of ongoing study and may provide insight into filarial host specificity.

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The Radiation-Induced Regenerative Response of Adult Tissue-Specific Stem Cells: Models and Signaling Pathways

Cancers ◽

10.3390/cancers13040855 ◽

2021 ◽

Vol 13 (4) ◽

pp. 855

Author(s):

Paola Serrano Martinez ◽

Lorena Giuranno ◽

Marc Vooijs ◽

Robert P. Coppes

Keyword(s):

Signaling Pathways ◽

Progenitor Cells ◽

Tissue Regeneration ◽

Normal Tissue ◽

Cellular Response ◽

Adult Tissue ◽

Tissue Specific ◽

Radiation Induced

Radiotherapy is involved in the treatment of many cancers, but damage induced to the surrounding normal tissue is often inevitable. Evidence suggests that the maintenance of homeostasis and regeneration of the normal tissue is driven by specific adult tissue stem/progenitor cells. These tasks involve the input from several signaling pathways. Irradiation also targets these stem/progenitor cells, triggering a cellular response aimed at achieving tissue regeneration. Here we discuss the currently used in vitro and in vivo models and the involved specific tissue stem/progenitor cell signaling pathways to study the response to irradiation. The combination of the use of complex in vitro models that offer high in vivo resemblance and lineage tracing models, which address organ complexity constitute potential tools for the study of the stem/progenitor cellular response post-irradiation. The Notch, Wnt, Hippo, Hedgehog, and autophagy signaling pathways have been found as crucial for driving stem/progenitor radiation-induced tissue regeneration. We review how these signaling pathways drive the response of solid tissue-specific stem/progenitor cells to radiotherapy and the used models to address this.

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Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility

Micromachines ◽

10.3390/mi12030346 ◽

2021 ◽

Vol 12 (3) ◽

pp. 346

Author(s):

Hui Ling Ma ◽

Ana Carolina Urbaczek ◽

Fayene Zeferino Ribeiro de Souza ◽

Paulo Augusto Gomes Garrido Carneiro Leão ◽

Janice Rodrigues Perussi ◽

...

Keyword(s):

Shear Stress ◽

Cell Viability ◽

Cellular Response ◽

Fluid Shear Stress ◽

Umbilical Vein ◽

Microfluidic Devices ◽

In Vitro Assays ◽

Stress Effect

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.

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Effects of Platelet-Rich Plasma on Cellular Populations of the Central Nervous System: The Influence of Donor Age

International Journal of Molecular Sciences ◽

10.3390/ijms22041725 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1725

Author(s):

Diego Delgado ◽

Ane Miren Bilbao ◽

Maider Beitia ◽

Ane Garate ◽

Pello Sánchez ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Cellular Response ◽

Platelet Rich Plasma ◽

Biological Response ◽

In Vitro Models ◽

Molecular Composition ◽

Cellular Models ◽

The Central Nervous System

Platelet-rich plasma (PRP) is a biologic therapy that promotes healing responses across multiple medical fields, including the central nervous system (CNS). The efficacy of this therapy depends on several factors such as the donor’s health status and age. This work aims to prove the effect of PRP on cellular models of the CNS, considering the differences between PRP from young and elderly donors. Two different PRP pools were prepared from donors 65–85 and 20–25 years old. The cellular and molecular composition of both PRPs were analyzed. Subsequently, the cellular response was evaluated in CNS in vitro models, studying proliferation, neurogenesis, synaptogenesis, and inflammation. While no differences in the cellular composition of PRPs were found, the molecular composition of the Young PRP showed lower levels of inflammatory molecules such as CCL-11, as well as the presence of other factors not found in Aged PRP (GDF-11). Although both PRPs had effects in terms of reducing neural progenitor cell apoptosis, stabilizing neuronal synapses, and decreasing inflammation in the microglia, the effect of the Young PRP was more pronounced. In conclusion, the molecular composition of the PRP, conditioned by the age of the donors, affects the magnitude of the biological response.

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The Interactome of Cancer-Related Lysyl Oxidase and Lysyl Oxidase-Like Proteins

Cancers ◽

10.3390/cancers13010071 ◽

2020 ◽

Vol 13 (1) ◽

pp. 71

Author(s):

Sylvain D. Vallet ◽

Coline Berthollier ◽

Romain Salza ◽

Laurent Muller ◽

Sylvie Ricard-Blum

Keyword(s):

Protein Folding ◽

Cancer Progression ◽

Cellular Response ◽

Lysyl Oxidase ◽

Chaperone Activity ◽

Cross Linking ◽

Binding Assays ◽

Vessel Development ◽

New Interactions

The members of the lysyl oxidase (LOX) family are amine oxidases, which initiate the covalent cross-linking of the extracellular matrix (ECM), regulate ECM stiffness, and contribute to cancer progression. The aim of this study was to build the first draft of the interactome of the five members of the LOX family in order to determine its molecular functions, the biological and signaling pathways mediating these functions, the biological processes it is involved in, and if and how it is rewired in cancer. In vitro binding assays, based on surface plasmon resonance and bio-layer interferometry, combined with queries of interaction databases and interaction datasets, were used to retrieve interaction data. The interactome was then analyzed using computational tools. We identified 31 new interactions and 14 new partners of LOXL2, including the α5β1 integrin, and built an interactome comprising 320 proteins, 5 glycosaminoglycans, and 399 interactions. This network participates in ECM organization, degradation and cross-linking, cell-ECM interactions mediated by non-integrin and integrin receptors, protein folding and chaperone activity, organ and blood vessel development, cellular response to stress, and signal transduction. We showed that this network is rewired in colorectal carcinoma, leading to a switch from ECM organization to protein folding and chaperone activity.

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The Role of Biomimetic Hypoxia on Cancer Cell Behaviour in 3D Models: A Systematic Review

Cancers ◽

10.3390/cancers13061334 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1334

Author(s):

Ye Liu ◽

Zahra Mohri ◽

Wissal Alsheikh ◽

Umber Cheema

Keyword(s):

Systematic Review ◽

Cellular Response ◽

3D Culture ◽

3D Models ◽

In Vitro Models ◽

Research Findings ◽

Tissue Models

The development of biomimetic, human tissue models is recognized as being an important step for transitioning in vitro research findings to the native in vivo response. Oftentimes, 2D models lack the necessary complexity to truly recapitulate cellular responses. The introduction of physiological features into 3D models informs us of how each component feature alters specific cellular response. We conducted a systematic review of research papers where the focus was the introduction of key biomimetic features into in vitro models of cancer, including 3D culture and hypoxia. We analysed outcomes from these and compiled our findings into distinct groupings to ascertain which biomimetic parameters correlated with specific responses. We found a number of biomimetic features which primed cancer cells to respond in a manner which matched in vivo response.

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Targeting of p38 Mitogen-Activated Protein Kinases to MEF2 Transcription Factors

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4028 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4028-4038 ◽

Cited By ~ 172

Author(s):

Shen-Hsi Yang ◽

Alex Galanis ◽

Andrew D. Sharrocks

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Cellular Response ◽

Map Kinases ◽

Biological Response ◽

Extracellular Signals ◽

Extracellular Signal ◽

Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38α and p38β2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).

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Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK

Biochemical Journal ◽

10.1042/bj3550297 ◽

2001 ◽

Vol 355 (2) ◽

pp. 297-305 ◽

Cited By ~ 25

Author(s):

Diana L. LEFEBVRE ◽

Yahong BAI ◽

Nazanin SHAHMOLKY ◽

Monika SHARMA ◽

Raymond POON ◽

...

Keyword(s):

Protein Kinase ◽

Cellular Response ◽

Catalytic Domain ◽

Metabolic Stress ◽

Glucose Deprivation ◽

Protein Band ◽

Northern Blotting ◽

Significant Similarity ◽

Amp Activated Protein Kinase

Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress.

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Studies of the interaction of ticagrelor with the P2Y13 receptor and with P2Y13-dependent pro-platelet formation by human megakaryocytes

Thrombosis and Haemostasis ◽

10.1160/th15-10-0829 ◽

2016 ◽

Vol 116 (12) ◽

pp. 1079-1088 ◽

Cited By ~ 9

Author(s):

Anna Björquist ◽

Christian A. Di Buduo ◽

Eti A. Femia ◽

Robert F. Storey ◽

Richard C. Becker ◽

...

Keyword(s):

Platelet Count ◽

Cellular Response ◽

P2y Receptors ◽

Cell System ◽

Experimental Models ◽

Significant Activity ◽

Label Free ◽

Coronary Syndrome ◽

Platelet Formation

SummaryTicagrelor is an antagonist of the platelet P2Y12 receptor for ADP, approved for the prevention of thromboembolic events in patients with acute coronary syndrome. Previous studies showed that ticagrelor has no significant activity versus P1 receptors for adenosine and other known P2Y receptors, with the exception of P2Y13, which was not tested. The P2Y12 antagonist cangrelor has been shown to also inhibit P2Y13 and to decrease the P2Y13-regulated capacity of megakaryocytes to produce pro-platelets. We tested whether or not ticagrelor inhibits P2Y13 signalling and function. The in vitro effects of ticagrelor, its active (TAM) and inactive (TIM) metabolites, cangrelor and the P2Y13 antagonist MRS2211 were tested in two experimental models: 1) a label-free cellular response assay in P2Y13-transfected HEK293 T-REx cells; and 2) pro-platelet formation by human megakaryocytes in culture. Ticagrelor, TAM, cangrelor and MRS2211, but not TIM, inhibited the cellular responses in P2Y13-transfected cells. In contrast, only MRS2211 and cangrelor, confirming previous results, inhibited pro-platelet formation by megakaryocytes in vitro. The platelet count of patients randomised to treatment with ticagrelor in the PLATO trial did not change during treatment and was comparable to those of patients randomised to clopidogrel. In conclusion, ticagrelor and TAM act as P2Y13 antagonists in a transfected cell system in vitro but this does not translate into any impact on pro-platelet formation in vitro or altered platelet count in patients.

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