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Journal of Extracellular Vesicles Jun 2023Purifying extracellular vesicles (EVs) has been challenging because EVs are heterogeneous in cargo yet share similar sizes and densities. Most surface marker-based...
Purifying extracellular vesicles (EVs) has been challenging because EVs are heterogeneous in cargo yet share similar sizes and densities. Most surface marker-based affinity separation methods are limited to research or diagnostic scales. We report that heparin chromatography can separate purified EVs into two distinct subpopulations as ascertained by MS/MS: a non-heparin-binding (NHB) fraction that contains classical EV markers such as tetraspanins and a heparin-binding (HB) fraction enriched in fibronectins and histones. Both fractions were similarly fusogenic but induced different transcriptional responses in endothelial cells. While EVs that were purified by conventional, non-affinity methods alone induced ERK1/2 phosphorylation and Ki67, the NHB fraction did not. This result suggests heparin chromatography as an additional novel fractionation step that is inherently scalable, does not lead to loss of material, and separates inflammatory and pyrogenic EVs from unreactive EVs, which will improve clinical applications.
Topics: Heparin; Tandem Mass Spectrometry; Endothelial Cells; Extracellular Vesicles; Chromatography, Affinity
PubMed: 37272197
DOI: 10.1002/jev2.12327 -
Toxins Mar 2019Staphylococcal enterotoxin B (SEB) and related superantigenic toxins produced by are potent activators of the immune system. These protein toxins bind to major... (Review)
Review
Staphylococcal enterotoxin B (SEB) and related superantigenic toxins produced by are potent activators of the immune system. These protein toxins bind to major histocompatibility complex (MHC) class II molecules and specific Vβ regions of T-cell receptors (TCRs), resulting in the activation of both monocytes/macrophages and T lymphocytes. The bridging of TCRs with MHC class II molecules by superantigens triggers an early "cytokine storm" and massive polyclonal T-cell proliferation. Proinflammatory cytokines, tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 elicit fever, inflammation, multiple organ injury, hypotension, and lethal shock. Upon MHC/TCR ligation, superantigens induce signaling pathways, including mitogen-activated protein kinase cascades and cytokine receptor signaling, which results in NFκB activation and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. In addition, gene profiling studies have revealed the essential roles of innate antimicrobial defense genes in the pathogenesis of SEB. The genes expressed in a murine model of SEB-induced shock include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, endoplasmic reticulum/mitochondrial stress responses, immunoproteasome components, and IFN-stimulated genes. This review focuses on the signaling pathways induced by superantigens that lead to the activation of inflammation and damage response genes. The induction of these damage response genes provides evidence that SEB induces danger signals in host cells, resulting in multiorgan injury and toxic shock. Therapeutics targeting both host inflammatory and cell death pathways can potentially mitigate the toxic effects of staphylococcal superantigens.
Topics: Animals; Bacterial Toxins; Cell Death; Cytokines; Humans; Oxidative Stress; Pyrogens; Receptors, Antigen, T-Cell; Shock, Septic; Signal Transduction; Staphylococcus; Superantigens
PubMed: 30909619
DOI: 10.3390/toxins11030178 -
ALTEX 2021Monocyte activation tests (MAT) are widely available but rarely used in place of animal-based pyrogen tests for safety assessment of medical devices. To address this...
Monocyte activation tests (MAT) are widely available but rarely used in place of animal-based pyrogen tests for safety assessment of medical devices. To address this issue, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods and the PETA International Science Consortium Ltd. convened a workshop at the National Institutes of Health on September 18-19, 2018. Participants included representatives from MAT testing laboratories, medical device manufacturers, the U.S. Food and Drug Administration's Center for Devices and Radiologic Health (CDRH), the U.S. Pharmacopeia, the International Organization for Standardization, and experts in the development of MAT protocols. Discussions covered industry experiences with the MAT, remaining challenges, and how CDRH's Medical Device Development Tools (MDDT) Program, which qualifies tools for use in evaluating medical devices to streamline device development and regulatory evaluation, could be a pathway to qualify the use of MAT in place of the rabbit pyrogen test and the limulus amebocyte lysate test for medical device testing. Workshop outcomes and follow-up activities are discussed.
Topics: Animal Testing Alternatives; Animals; Endotoxins; Equipment and Supplies; Monocytes; Pyrogens; Rabbits; Toxicity Tests
PubMed: 33452530
DOI: 10.14573/altex.2012021 -
Frontiers in Cellular Neuroscience 2015Inflammation is the key host-defense response to infection and injury, yet also a major contributor to a diverse range of diseases, both peripheral and central in... (Review)
Review
Inflammation is the key host-defense response to infection and injury, yet also a major contributor to a diverse range of diseases, both peripheral and central in origin. Brain injury as a result of stroke or trauma is a leading cause of death and disability worldwide, yet there are no effective treatments, resulting in enormous social and economic costs. Increasing evidence, both preclinical and clinical, highlights inflammation as an important factor in stroke, both in determining outcome and as a contributor to risk. A number of inflammatory mediators have been proposed as key targets for intervention to reduce the burden of stroke, several reaching clinical trial, but as yet yielding no success. Many factors could explain these failures, including the lack of robust preclinical evidence and poorly designed clinical trials, in addition to the complex nature of the clinical condition. Lack of consideration in preclinical studies of associated co-morbidities prevalent in the clinical stroke population is now seen as an important omission in previous work. These co-morbidities (atherosclerosis, hypertension, diabetes, infection) have a strong inflammatory component, supporting the need for greater understanding of how inflammation contributes to acute brain injury. Interleukin (IL)-1 is the prototypical pro-inflammatory cytokine, first identified many years ago as the endogenous pyrogen. Research over the last 20 years or so reveals that IL-1 is an important mediator of neuronal injury and blocking the actions of IL-1 is beneficial in a number of experimental models of brain damage. Mechanisms underlying the actions of IL-1 in brain injury remain unclear, though increasing evidence indicates the cerebrovasculature as a key target. Recent literature supporting this and other aspects of how IL-1 and systemic inflammation in general contribute to acute brain injury are discussed in this review.
PubMed: 25705177
DOI: 10.3389/fncel.2015.00018 -
Scientific Reports Aug 2023Intrinsic or added immune activating molecules are key for most vaccines to provide desired immunity profiles but may increase systemic reactogenicity. Regulatory...
Intrinsic or added immune activating molecules are key for most vaccines to provide desired immunity profiles but may increase systemic reactogenicity. Regulatory agencies require rabbit pyrogen testing (RPT) for demonstration of vaccine reactogenicity. Recently, the monocyte activation test (MAT) gained popularity as in vitro alternative, yet this assay was primarily designed to test pyrogen-free products. The aim was to adjust the MAT to enable testing of pyrogen containing vaccines in an early stage of development where no reference batch is yet available. The MAT and RPT were compared for assessing unknown safety profiles of pertussis outer membrane vesicle (OMV) vaccine candidates to those of Bexsero as surrogate reference vaccine. Pertussis OMVs with wild-type LPS predominantly activated TLR2 and TLR4 and were more reactogenic than Bexsero. However, this reactogenicity profile for pertussis OMVs could be equalized or drastically reduced compared to Bexsero or a whole-cell pertussis vaccine, respectively by dose changing, modifying the LPS, intranasal administration, or a combination of these. Importantly, except for LPS modified products, reactogenicity profiles obtained with the RPT and MAT were comparable. Overall, we demonstrated that this pertussis OMV vaccine candidate has an acceptable safety profile. Furthermore, the MAT proved its applicability to assess reactogenicity levels of pyrogen containing vaccines at multiple stages of vaccine development and could eventually replace rabbit pyrogen testing.
Topics: Animals; Rabbits; Lipopolysaccharides; Whooping Cough; Pyrogens; Monocytes; Biological Assay
PubMed: 37542099
DOI: 10.1038/s41598-023-39908-7 -
Frontiers in Immunology 2019The pyrogenic property being the first activity described, members of the interleukin-1 superfamily (IL-1α, IL-1β, IL-18, and the newest members: IL-33, IL-36, IL-37,... (Review)
Review
The pyrogenic property being the first activity described, members of the interleukin-1 superfamily (IL-1α, IL-1β, IL-18, and the newest members: IL-33, IL-36, IL-37, and IL-38) are now known to be involved in several inflammatory diseases such as obesity, atherosclerosis, cancer, viral and parasite infections, and auto-inflammatory syndromes as well as liver diseases. Inflammation processes are keystones of chronic liver diseases, of which the etiology may be viral or toxic, as in alcoholic or non-alcoholic liver diseases. Inflammation is also at stake in acute liver failure involving massive necrosis, and in ischemia-reperfusion injury in the setting of liver transplantation. The role of the IL-1 superfamily of cytokines and receptors in liver diseases can be either protective or pro-inflammatory, depending on timing and the environment. Our review provides an overview of current understanding of the IL-1 family members in liver inflammation, highlighting recent key investigations, and therapeutic perspectives. We have tried to apply the concept of trained immunity to liver diseases, based on the role of the members of the IL-1 superfamily, first of all IL-1β but also IL-18 and IL-33, in modulating innate lymphoid immunity carried by natural killer cells, innate lymphoid cells or innate T-αβ lymphocytes.
Topics: Animals; Cytokines; Disease Susceptibility; Genetic Predisposition to Disease; Hepatitis; Humans; Interleukin-1; Liver Diseases; Multigene Family; Receptors, Interleukin-1
PubMed: 31507607
DOI: 10.3389/fimmu.2019.02014 -
Journal of Biomedical Science Jul 2023Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist...
BACKGROUND
Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase.
METHODS
Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry.
RESULTS
Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation.
CONCLUSIONS
Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.
Topics: Streptococcus pyogenes; Neutrophils; Bacterial Proteins; Exotoxins
PubMed: 37430325
DOI: 10.1186/s12929-023-00947-x -
Scientific Reports Jul 2022Many medical procedures could benefit from the use of tissue sealants which allow for reduced surgery time, limited blood loss, easier tissue handling, and fewer...
Many medical procedures could benefit from the use of tissue sealants which allow for reduced surgery time, limited blood loss, easier tissue handling, and fewer postoperative complications. The safety and biocompatibility of surgical sealants are of paramount importance therefore, the aim of this study is to investigate the biocompatibility of NE'X Glue Surgical Adhesive. Chemical characterization (VOC and elements), cytotoxicity (MEM elution), genotoxicity (AMES and MLA), endotoxin contamination, sensitization potential, intracutaneous reactivity, acute and subchronic systemic toxicity with implantation as well as pyrogenicity were evaluated to investigate the biocompatibility of the NE'X Glue Surgical Adhesive. Studies were conducted according to ISO 10993 standards. The biocompatibility requirements with accordance to ISO 10993-1 for NE'X Glue were met. In vitro studies showed that NE'X Glue surgical adhesive is non-cytotoxic and non-mutagenic. Also, in vivo studies demonstrated that NE'X Glue shows no signs of toxicity, has no pyrogenic potential, and is non-sensitizing and non-irritating. The chemical characterization showed that no compounds were identified above Analytical Evaluation Threshold (AET), and no elements with concentrations higher than element-specific PDE (µg/day) were detected. NE'X Glue Surgical Adhesive is a versatile and promising new surgical sealant with a wide range of potential applications and very good*0* biocompatibility.
Topics: Albumins; Aldehydes; Humans; Injections; Postoperative Complications; Tissue Adhesives
PubMed: 35882896
DOI: 10.1038/s41598-022-16853-5 -
Journal of Maxillofacial and Oral... Jun 2015The aim of this paper is to review the pathophysiology of thermoregulation mechanism, various causes of fever after maxillofacial surgery and the different treatment... (Review)
Review
PURPOSE
The aim of this paper is to review the pathophysiology of thermoregulation mechanism, various causes of fever after maxillofacial surgery and the different treatment protocols advised in the literature.
DISCUSSION
Fever is one of the most common complaints after major surgery and is also considered to be an important clinical sign which indicates developing pathology that may go unnoticed by the clinician during post operative period. Several factors are responsible for fever after the maxillofacial surgery, inflammation and infection being the commonest. However, other rare causes such as drug allergy, dehydration, malignancy and endocrinological disorders, etc. should be ruled out prior to any definite diagnosis and initiate the treatment. Proper history and clinical examination is an essential tool to predict the causative factors for fever. Common cooling methods like tepid sponging are usually effective alone or in conjunction with analgesics to reduce the temperature.
CONCLUSION
Fever is a common postoperative complaint and should not be underestimated as it may indicate a more serious underlying pathology. A specific guideline towards the management of such patients is necessary in every hospital setting to ensure optimal care towards the patients during post operative period.
PubMed: 26028829
DOI: 10.1007/s12663-013-0611-7