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1.
Front Immunol ; 15: 1363185, 2024.
Article in English | MEDLINE | ID: mdl-38660297

ABSTRACT

Extracellular vesicles (EVs) have important roles as mediators of cell-to-cell communication, with physiological functions demonstrated in various in vivo models. Despite advances in our understanding of the biological function of EVs and their potential for use as therapeutics, there are limitations to the clinical approaches for which EVs would be effective. A primary determinant of the biodistribution of EVs is the profile of proteins and other factors on the surface of EVs that define the tropism of EVs in vivo. For example, proteins displayed on the surface of EVs can vary in composition by cell source of the EVs and the microenvironment into which EVs are delivered. In addition, interactions between EVs and recipient cells that determine uptake and endosomal escape in recipient cells affect overall systemic biodistribution. In this review, we discuss the contribution of the EV donor cell and the role of the microenvironment in determining EV tropism and thereby determining the uptake and biological activity of EVs.


Subject(s)
Extracellular Vesicles , Extracellular Vesicles/metabolism , Humans , Animals , Cell Communication , Cellular Microenvironment
2.
Mol Ther ; 2024 Feb 19.
Article in English | MEDLINE | ID: mdl-38379282

ABSTRACT

Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads to coordinate the response of multiple cell types in cutaneous wound healing. Here we used a cutaneous injury model as a donor of pro-reparative EVs to treat recipient diabetic obese mice, a model of impaired wound healing. We established a functional screen for microRNAs (miRNAs) that increased the pro-reparative activity of EVs and identified a down-regulation of miR-425-5p in EVs in vivo and in vitro associated with the regulation of adiponectin. We tested a cell type-specific reporter of a tetraspanin CD9 fusion with GFP to lineage map the release of EVs from macrophages in the wound bed, based on the expression of miR-425-5p in macrophage-derived EVs and the abundance of macrophages in EV donor sites. Analysis of different promoters demonstrated that EV release under the control of a macrophage-specific promoter was most abundant and that these EVs were internalized by dermal fibroblasts. These findings suggested that pro-reparative EVs deliver miRNAs, such as miR-425-5p, that stimulate the expression of adiponectin that has insulin-sensitizing properties. We propose that EVs promote intercellular signaling between cell layers in the skin to resolve inflammation, induce proliferation of basal keratinocytes, and accelerate wound closure.

3.
J Trauma Acute Care Surg ; 96(3): 371-377, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-37880828

ABSTRACT

BACKGROUND: Acute lung injury and subsequent resolution following severe injury are coordinated by a complex lung microenvironment that includes extracellular vesicles (EVs). We hypothesized that there is a heterogenous population of EVs recruited to the alveoli postinjury and that we could identify specific immune-relevant mediators expressed on bronchoalveolar lavage (BAL) EVs as candidate biomarkers of injury and injury resolution. METHODS: Mice underwent 30% TBSA burn injury and BAL fluid was collected 4 hours postinjury and compared with sham. Extracellular vesicles were purified and single vesicle flow cytometry (vFC) was performed using fluorescent antibodies to quantify the expression of specific cell surface markers on individual EVs. Next, we evaluated human BAL specimens from injured patients to establish translational relevance of the mouse vFC analysis. Human BAL was collected from intubated patients following trauma or burn injury, EVs were purified, then subjected to vFC analysis. RESULTS: A diverse population of EVs were mobilized to the alveoli after burn injury in mice. Quantitative BAL vFC identified significant increases in macrophage-derived CD44+ EVs (preinjury, 10.8% vs. postinjury, 13%; p < 0.05) and decreases in IL-6 receptor alpha (CD126) EVs (preinjury, 19.3% vs. postinjury, 9.3%, p < 0.05). Bronchoalveolar lavage from injured patients also contained a heterogeneous population of EVs derived from myeloid cells, endothelium, and epithelium sources, with CD44+ EVs being highly detected. CONCLUSION: Injury causes mobilization of a heterogeneous population of EVs to the alveoli in both animal models and injured patients. Defining EV release after injury will be critical in identifying diagnostic and therapeutic targets to limit postinjury acute lung injury.


Subject(s)
Acute Lung Injury , Extracellular Vesicles , Humans , Animals , Mice , Lung , Extracellular Vesicles/metabolism , Acute Lung Injury/therapy , Pulmonary Alveoli , Bronchoalveolar Lavage Fluid
4.
J Nanobiotechnology ; 20(1): 474, 2022 Nov 05.
Article in English | MEDLINE | ID: mdl-36335351

ABSTRACT

Chronic metabolic diseases such as diabetes are characterized by delayed wound healing and a dysregulation of the inflammatory phase of wound repair. Our study focuses on changes in the payload of extracellular vesicles (EVs) communicating between immune cells and stromal cells in the wound bed, which regulate the rate of wound closure. Adoptive transfer of EVs from genetically defined mouse models are used here to demonstrate a functional and molecular basis for differences in the pro-reparative biological activity of diabetic (db/db) vs. wildtype EVs in wound healing. We identify several members of the Serpin family of serine protease inhibitors that are absent in db/db EVs, then we overexpress Serpin A1, F2 and G1 in EVs to evaluate their effect on wound healing in db/db mice. Serpins have an important role in regulating levels of elastase, plasmin and complement factors that coordinate immune cell signaling in full thickness wounds in a diabetic model. Here, we establish a novel therapeutic approach by engineering the payload of EVs based on proteomic analysis. Serpin-loaded EVs were used to rescue the Serpin deficiency identified by proteomics and promote wound healing in db/db mice, as well as evaluated how EVs affected extracellular matrix remodeling and the resolution of tissue injury. Therefore, we propose that the identification of EV payloads that are downregulated in diabetic wounds can be systematically analyzed for their functional activity and potential as a therapeutic, based on whether their re-expression in engineered EVs restores normal kinetics of tissue repair in chronic wounds.


Subject(s)
Diabetes Mellitus , Extracellular Vesicles , Serpins , Mice , Animals , Serpins/pharmacology , Proteomics , Wound Healing , Disease Models, Animal
5.
Front Genet ; 13: 932555, 2022.
Article in English | MEDLINE | ID: mdl-36092940

ABSTRACT

Otitis media (OM), the most common disease of childhood, is typically characterized by bacterial infection of the middle ear (ME). Prominent features of OM include hyperplasia of the ME mucosa, which transforms from a monolayer of simple squamous epithelium with minimal stroma into a full-thickness respiratory epithelium in 2-3 days after infection. Analysis of the murine ME transcriptome during OM showed down-regulation of the tumor suppressor gene Ecrg4 that was temporally related to mucosal hyperplasia and identified stromal cells as the primary ECRG4 source. The reduction in Ecrg4 gene expression coincided with the cleavage of ECRG4 protein to release an extracellular fragment, augurin. The duration of mucosal hyperplasia during OM was greater in Ecrg4 -/- mice, the number of infiltrating macrophages was enhanced, and ME infection cleared more rapidly. ECRG4-null macrophages showed increased bacterial phagocytosis. Co-immunoprecipitation identified an association of augurin with TLR4, CD14 and MD2, the components of the lipopolysaccharide (LPS) receptor. The results suggest that full-length ECRG4 is a sentinel molecule that potentially inhibits growth of the ME stroma. Processing of ECRG4 protein during inflammation, coupled with a decline in Ecrg4 gene expression, also influences the behavior of cells that do not express the gene, limiting the production of growth factors by epithelial and endothelial cells, as well as the activity of macrophages.

6.
J Trauma Acute Care Surg ; 93(1): 38-42, 2022 07 01.
Article in English | MEDLINE | ID: mdl-35727591

ABSTRACT

BACKGROUND: Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in trauma patients worldwide. Brain injury is associated with significant inflammation, both within the brain and in the peripheral organs. This inflammatory response in TBI leads to a secondary injury, worsening the effects of the original brain injury. Serotonin is also linked to inflammation in the intestine and inflammatory bowel disease, but its role in the gut-brain axis is not known. We hypothesized that using fluoxetine to block serotonin reuptake would reduce organ inflammation and improve outcomes after TBI. METHODS: C57/B6 mice were given a severe TBI using a controlled cortical impact. To measure intestinal permeability, a piece of terminal ileum was resected, the lumen was filled with 4-kDa fluorescein isothiocyanate (FITC)-dextran, and the ends were tied. The intestinal segment was submerged in buffer and fluorescence in the buffer measured over time. To measure lung permeability, 70-kDa FITC-dextran is injected retro-orbitally. Thirty minutes later, the left lung was homogenized and the fluorescence was measured. To measure performance on the rota-rod, mice were placed on a spinning rod, and the time to fall off was measured. Those treated with fluoxetine received a single dose of 5 mg/kg via intraperitoneal injection immediately after injury. RESULTS: Traumatic brain injury was associated with an increase in intestinal permeability to FITC-dextran, increased lung vascular permeability, and worse performance on the rota-rod. Fluoxetine significantly reduced lung and intestinal permeability after TBI and improved performance on the rota-rod after TBI. CONCLUSION: Use of fluoxetine has the potential to reduce lung injury and improve motor coordination in severe TBI patients. Further study will be needed to elucidate the mechanism behind this effect.


Subject(s)
Brain Injuries, Traumatic , Brain Injuries , Animals , Brain Injuries/complications , Brain Injuries, Traumatic/complications , Brain Injuries, Traumatic/drug therapy , Disease Models, Animal , Fluoxetine/pharmacology , Fluoxetine/therapeutic use , Humans , Inflammation/complications , Mice , Serotonin/therapeutic use
7.
J Trauma Acute Care Surg ; 92(2): 323-329, 2022 02 01.
Article in English | MEDLINE | ID: mdl-34789702

ABSTRACT

BACKGROUND: The systemic inflammatory response (SIRS) drives late morbidity and mortality after injury. The α7 nicotinic acetylcholine receptor (α7nAchR) expressed on immune cells regulates the vagal anti-inflammatory pathway that prevents an overwhelming SIRS response to injury. Nonspecific pharmacologic stimulation of the vagus nerve has been evaluated as a potential therapeutic to limit SIRS. Unfortunately, the results of clinical trials have been underwhelming. We hypothesized that directly targeting the α7nAchR would more precisely stimulate the vagal anti-inflammatory pathway on immune cells and decrease gut and lung injury after severe burn. METHODS: C57BL/6 mice underwent 30% total body surface area steam burn. Mice were treated with an intraperitoneal injection of a selective agonist of the α7nAchR (AR-R17779) at 30 minutes postburn. Intestinal permeability to 4 kDa FITC-dextran was measured at multiple time points postinjury. Lung vascular permeability was measured 6 hours after burn injury. Serial behavioral assessments were performed to quantify activity levels. RESULTS: Intestinal permeability peaked at 6 hours postburn. AR-R17779 decreased burn-induced intestinal permeability in a dose-dependent fashion (p < 0.001). There was no difference in gut permeability to 4 kDa FITC-dextran between sham and burn-injured animals treated with 5 mg/kg of AR-R17779. While burn injury increased lung permeability 10-fold, AR-R17779 prevented burn-induced lung permeability with no difference compared with sham (p < 0.01). Postinjury activity levels were significantly improved in burned animals treated with AR-R17779. CONCLUSION: Directly stimulating the α7nAchR prevents burn-induced gut and lung injury. Directly targeting the α7nAChR that mediates the cholinergic anti-inflammatory response may be an improved strategy compared with nonspecific vagal agonists.


Subject(s)
Burns/complications , Neuroimmunomodulation , Systemic Inflammatory Response Syndrome/etiology , Systemic Inflammatory Response Syndrome/prevention & control , Vagus Nerve/drug effects , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Animals , Dextrans/pharmacology , Disease Models, Animal , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/pharmacology , Intestinal Mucosa/metabolism , Lung Injury/metabolism , Male , Mice , Mice, Inbred C57BL , Permeability
8.
Inflamm Res ; 71(1): 9-11, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34792616

ABSTRACT

INTRODUCTION: The CHRNA7 gene encodes the α-7 nicotinic acetylcholine receptor (α7nAchR) that regulates anti-inflammatory responses to injury; however, only humans express a variant gene called CHRFAM7A that alters the function of α7nAChR; CHRFAM7A expression predominates in bone marrow and monocytes/macrophages where the CHRFAM7A/CHRNA7 ratio is highly variable between individuals. We have previously shown in transgenic mice that CHRFAM7A increased emergency myelopoiesis from the bone marrow and monocyte/macrophage expression in lungs. MATERIALS AND METHODS: CHRFAM7A transgenic mice are compared to age- and gender-matched wild-type (WT) siblings. We utilized a model of sepsis using LPS injection to measure survival. Lung vascular permeability was measured after severe burn injury in WT vs. CHRFAM7A transgenic mice. Bone marrow CHRFAM7A expression was evaluated using adoptive transfer of CHRFAM7A transgenic bone marrow into WT mice. RESULTS: Here, we demonstrate that CHRFAM7A expression results in an anti-inflammatory phenotype with an improved survival to LPS and decreased acute lung injury in a severe cutaneous burn model compared to WT. CONCLUSIONS: These data suggest that the relative expression of CHRFAM7A may alter resiliency to injury and contribute to individual variability in the human systemic inflammatory response (SIRS) to injury.


Subject(s)
Leukocytes , alpha7 Nicotinic Acetylcholine Receptor , Animals , Anti-Inflammatory Agents , Mice , Mice, Transgenic , alpha7 Nicotinic Acetylcholine Receptor/metabolism
9.
Nanomedicine ; 38: 102447, 2021 11.
Article in English | MEDLINE | ID: mdl-34314868

ABSTRACT

Therapeutics based on stem cell technology, including stem cell-derived exosomes, have emerged in recent years for the treatment of what were otherwise considered incurable diseases. In this study, we evaluated the efficacy of human MSC-derived exosomes for protection against cisplatin induced ototoxic hearing loss. Incubation of cochlear explants with MSC-derived exosomes prior to addition of cisplatin induced a reduction in cisplatin-induced drug toxicity in auditory hair cells but not when the exosomes were introduced simultaneously with or after cisplatin. The delivery of MSC-derived exosomes to cochlear explants was confirmed by the increasing protein levels of the exosome markers CD63 and HSP70 to reduce apoptosis. These results were consistent with those from a model in which MSC-derived exosomes protect auditory hair cells from cisplatin-induced drug toxicity in an ex vivo cochlear explant model and support future studies into the therapeutic benefits of stem cell-derived exosomes in clinical applications.


Subject(s)
Exosomes , Mesenchymal Stem Cells , Apoptosis , Cisplatin/adverse effects , Cisplatin/metabolism , Exosomes/metabolism , HSP70 Heat-Shock Proteins/metabolism , Humans , Mesenchymal Stem Cells/metabolism
10.
J Surg Res ; 268: 168-173, 2021 12.
Article in English | MEDLINE | ID: mdl-34314883

ABSTRACT

BACKGROUND: Extracellular vehicles (EVs) released by malignant tumor cells can mediate the immune response and promote metastasis through intercellular communication. EV analysis is an emerging cancer surveillance tool with advantages over traditional liquid biopsy methods. The aim of this pilot study is to identify actionable EV signatures in metastatic breast cancer. MATERIALS AND METHODS: Under an IRB-approved protocol for the analysis of patient plasma, samples were collected from women with newly diagnosed or progressive metastatic breast cancer and from women without cancer. Enriched EVs were analyzed via a bead-based multiplex assay designed to detect 37 distinct tumor-relevant epitopes. The mean fluorescent intensity of EV epitopes meeting a minimum threshold of detectability was compared between groups via independent samples t-test. Subgroup analysis was conducted for metastatic breast cancer patients who were positive for estrogen and/or progesterone receptors and negative for HER2. Other variables potentially affecting CD105 levels were also analyzed. RESULTS: CD105 was found to have a significantly higher mean fluorescent intensity in participants with metastatic breast cancer compared to control participants (P = 0.04). ER/PR+ subgroup analysis revealed a similar pattern compared to control participants (P = 0.01). Other analyzed variables were not found to have a significant correlation with CD105 levels. CONCLUSIONS: CD105 EV levels were significantly higher in samples from participants with breast cancer compared to controls. Given that CD105 is known to mediate angiogenesis and promote metastasis, EV-associated CD105 in plasma represents a potential biomarker for diagnosis, surveillance and therapeutic targeting in patients with metastatic breast cancer.


Subject(s)
Breast Neoplasms , Extracellular Vesicles , Biomarkers , Biomarkers, Tumor , Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Extracellular Vesicles/pathology , Female , Humans , Pilot Projects , Receptors, Progesterone
11.
Int J Mol Sci ; 22(7)2021 Mar 29.
Article in English | MEDLINE | ID: mdl-33805585

ABSTRACT

Extracellular vesicles (EVs) have an important role in mediating intercellular signaling in inflammation and affect the kinetics of wound healing, however, an understanding of the mechanisms regulating these responses remains limited. Therefore, we have focused on the use of cutaneous injury models in which to study the biology of EVs on the inflammatory phase of wound healing. For this, the foreign body response using sterile subcutaneous polyvinylalcohol (PVA) sponges is ideally suited for the parallel analysis of immune cells and EVs without the need for tissue dissociation, which would introduce additional variables. We have previously used this model to identify mediators of EV biogenesis, establishing that control of how EVs are made affects their payload and biological activity. These studies in normal mice led us to consider how conditions such as immunodeficiency and obsesity affect the profile of immune cells and EVs in this model using genetically defined mutant mice. Since EVs are intrinsically heterogenous in biological fluids, we have focused our studies on a novel technology, vesicle flow cytometry (vFC) to quantify changes in EVs in mouse models. Here, we show that myeloid-derived immune cells and EVs express proteins relevant in antigen presentation in PVA sponge implants that have distinct profiles in wildtype, immune-deficient (NOD scid) vs. diabetic (Leprdb) mice. Together, these results establish a foundation for the parallel analysis of both immune cells and EVs with technologies that begin to address the heterogeneity of intercellular communication in the wound bed.


Subject(s)
Antigens, CD/immunology , Extracellular Vesicles/physiology , Skin/injuries , Skin/pathology , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Diabetes Mellitus, Experimental/immunology , Disease Models, Animal , Extracellular Vesicles/immunology , Extracellular Vesicles/pathology , Kinetics , Male , Mice, Inbred C57BL , Mice, Inbred NOD/genetics , Mice, Inbred NOD/immunology , Mice, Obese/immunology , Myeloid Cells/immunology , Polyvinyl Alcohol , Wound Healing/immunology , Wound Healing/physiology
12.
Nat Commun ; 11(1): 6330, 2020 12 10.
Article in English | MEDLINE | ID: mdl-33303737

ABSTRACT

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.


Subject(s)
Neovascularization, Physiologic , Proteins/metabolism , Stress, Physiological , Animals , Cattle , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Endoplasmic Reticulum Chaperone BiP , Enzyme Activation/drug effects , Female , Glycosylation/drug effects , Heat-Shock Proteins/metabolism , Hexosamines/pharmacology , Hindlimb/pathology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Ischemia/pathology , MAP Kinase Signaling System/drug effects , Mice, Inbred C57BL , Microvessels/metabolism , Neovascularization, Physiologic/drug effects , Regional Blood Flow/drug effects , Signal Transduction/drug effects , Skin/pathology , Stress, Physiological/drug effects , Transcription Factor CHOP/metabolism , Unfolded Protein Response/drug effects , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-2/metabolism , Wound Healing/drug effects
13.
Inflamm Res ; 69(7): 631-633, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32303780

ABSTRACT

OBJECTIVE AND DESIGN: CHRFAM7A is a unique human gene that encodes a dominant negative inhibitor of the α7 nicotinic acetylcholine receptor. We have recently shown that CHRFAM7A is expressed in human leukocytes, increases cel-cell adhesion, and regulates the expression of genes associated with leukocyte migration. MATERIAL: Human THP-1, RAW264.7 and HEK293 cells. METHODS: Cell migration, cell proliferation and colony formation in soft agar to compare the biological activity of vector vs. CHRFAM7A-transduced cells. RESULTS: We show that gene delivery of CHRFAM7A into the THP-1 human monocytic cell line reduces cell migration, reduces chemotaxis to monocyte chemoattractant protein, and reduces colony formation in soft agar. CONCLUSION: Taken together, the findings demonstrate that CHRFAM7A regulates the biological activity of monocytes/macrophages to migrate and undergo anchorage-independent growth in vitro.


Subject(s)
alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors , Animals , Cell Adhesion/physiology , Cell Movement/genetics , Cell Movement/physiology , Cell Proliferation/physiology , Gene Expression , Gene Expression Regulation , HEK293 Cells , Humans , Leukocytes , Macrophages/physiology , Mice , Monocytes/physiology , RAW 264.7 Cells , Stem Cells/physiology , THP-1 Cells , Transduction, Genetic , alpha7 Nicotinic Acetylcholine Receptor/genetics , alpha7 Nicotinic Acetylcholine Receptor/physiology
14.
Sci Adv ; 6(11): eaay0518, 2020 03.
Article in English | MEDLINE | ID: mdl-32195341

ABSTRACT

The complex molecular microenvironment of the wound bed regulates the duration and degree of inflammation in the wound repair process, while its dysregulation leads to impaired healing. Understanding factors controlling this response provides therapeutic targets for inflammatory disease. Esophageal cancer-related gene 4 (ECRG4) is a candidate chemokine that is highly expressed on leukocytes. We used ECRG4 knockout (KO) mice to establish that the absence of ECRG4 leads to defective neutrophil recruitment with a delay in wound healing. An in vitro human promyelocyte model identified an ECRG4-mediated suppression of the hyaluronic acid receptor, CD44, a key receptor mediating inflammation resolution. In ECRG4 KO mouse leukocytes, there was an increase in CD44 expression, consistent with a model in which ECRG4 negatively regulates CD44 levels. Therefore, we propose a previously unidentified mechanism in which ECRG4 regulates early neutrophil recruitment and subsequent CD44-mediated resolution of inflammation.


Subject(s)
Gene Expression Regulation , Hyaluronan Receptors/biosynthesis , Neoplasm Proteins/metabolism , Neutrophils/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Humans , Hyaluronan Receptors/genetics , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Mice , Mice, Knockout , Neoplasm Proteins/genetics , Neutrophils/pathology , Tumor Suppressor Proteins/genetics
15.
Front Immunol ; 11: 606859, 2020.
Article in English | MEDLINE | ID: mdl-33391275

ABSTRACT

Tumor-derived extracellular vesicles (TEVs) are important regulators of the immune response in cancer; however, most research so far has been carried out using cell culture systems. Immune-competent murine tumor models currently provide the best platform to assess proposed roles of TEVs using in vivo animal models and therefore are important for examining interactions between TEVs and the immune system. In this review, we present the current knowledge on TEVs using in vivo tumor-bearing animal models, with a focus on the role of TEVs in mediating crosstalk between tumor cells and both adaptive and innate immune cells. In particular, we address the question how animal models can clarify the reported heterogeneity of TEV effects in both anti-tumor responses and evasion of immune surveillance. The potential of TEVs in mediating direct antigen-presenting functions supports their potential as cancer vaccine therapeutics, therefore, we provide an overview of key findings of TEV trials that have the potential as novel immunotherapies, and shed light on challenges in the path toward the first in-human trials. We also highlight the important updates on the methods that continue to enhance the rigor and reproducibility of EV studies, particularly in functional animal models.


Subject(s)
Extracellular Vesicles/immunology , Neoplasms, Experimental/immunology , Tumor Microenvironment , Adaptive Immunity , Animals , Extracellular Vesicles/metabolism , Extracellular Vesicles/pathology , Humans , Immunity, Innate , Immunotherapy , Mice , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neoplasms, Experimental/therapy , Signal Transduction , Tumor Escape
16.
Cell Mol Life Sci ; 76(24): 5027-5039, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31190084

ABSTRACT

The embedding of small peptide ligands within large inactive pre-pro-precursor proteins encoded by orphan open reading frames (ORFs) makes them difficult to identify and study. To address this problem, we generated oligonucleotide (< 100-400 base pair) combinatorial libraries from either the epidermal growth factor (EGF) ORF that encodes the > 1200 amino acid EGF precursor protein or the orphan ECRG4 ORF, that encodes a 148 amino acid Esophageal Cancer Related Gene 4 (ECRG4), a putative cytokine precursor protein of up to eight ligands. After phage display and 3-4 rounds of biopanning for phage internalization into prostate cancer epithelial cells, sequencing identified the 53-amino acid EGF ligand encoded by the 5' region of the EGF ORF and three distinct domains within the primary sequence of ECRG4: its membrane targeting hydrophobic signal peptide, an unanticipated amino terminus domain at ECRG437-63 and a C-terminus ECRG4133-148 domain. Using HEK-blue cells transfected with the innate immunity receptor complex, we show that both ECRG437-63 and ECRG4133-148 enter cells by interaction with the TLR4 immune complex but neither stimulate NFkB. Taken together, the results help establish that phage display can be used to identify cryptic domains within ORFs of the human secretome and identify a novel TLR4-targeted internalization domain in the amino terminus of ECRG4 that may contribute to its effects on cell migration, immune cell activation and tumor suppression.


Subject(s)
Immunity, Innate/genetics , Prostatic Neoplasms/genetics , Toll-Like Receptor 4/genetics , Tumor Suppressor Proteins/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Surface Display Techniques , Genes, Tumor Suppressor , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Male , NF-kappa B/genetics , Oligonucleotides/genetics , Open Reading Frames/genetics , Prostatic Neoplasms/pathology , Protein Domains/genetics , Transfection
17.
Anesthesiology ; 131(1): 132-147, 2019 07.
Article in English | MEDLINE | ID: mdl-31225809

ABSTRACT

BACKGROUND: As the meningeally derived, fibroblast-rich, mass-produced by intrathecal morphine infusion is not produced by all opiates, but reduced by mast cell stabilizers, the authors hypothesized a role for meningeal mast cell/fibroblast activation. Using the guinea pig, the authors asked: (1) Are intrathecal morphine masses blocked by opiate antagonism?; (2) Do opioid agonists not producing mast cell degranulation or fibroblast activation produce masses?; and (3) Do masses covary with Mas-related G protein-coupled receptor signaling thought to mediate mast cell degranulation? METHODS: In adult male guinea pigs (N = 66), lumbar intrathecal catheters connected to osmotic minipumps (14 days; 0.5 µl/h) were placed to deliver saline or equianalgesic concentrations of morphine sulfate (33 nmol/h), 2',6'-dimethyl tyrosine-(Tyr-D-Arg-Phe-Lys-NH2) (abbreviated as DMT-DALDA; 10 pmol/h; µ agonist) or PZM21 (27 nmol/h; biased µ agonist). A second pump delivered subcutaneous naltrexone (25 µg/h) in some animals. After 14 to 16 days, animals were anesthetized and perfusion-fixed. Drug effects on degranulation of human cultured mast cells, mouse embryonic fibroblast activation/migration/collagen formation, and Mas-related G protein-coupled receptor activation (PRESTO-Tango assays) were determined. RESULTS: Intrathecal infusion of morphine, DMT-DALDA or PZM21, but not saline, comparably increased thermal thresholds for 7 days. Spinal masses proximal to catheter tip, composed of fibroblast/collagen type I (median: interquartile range, 0 to 4 scale), were produced by morphine (2.3: 2.0 to 3.5) and morphine plus naltrexone (2.5: 1.4 to 3.1), but not vehicle (1.2: 1.1 to 1.5), DMT-DALDA (1.0: 0.6 to 1.3), or PZM21 (0.5: 0.4 to 0.8). Morphine in a naloxone-insensitive fashion, but not PZM21 or DMT-DALDA, resulted in mast cell degranulation and fibroblast proliferation/collagen formation. Morphine-induced fibroblast proliferation, as mast cell degranulation, is blocked by cromolyn. Mas-related G protein-coupled receptor activation was produced by morphine and TAN67 (∂-opioid agonist), but not by PZM21, TRV130 (mu biased ligand), or DMT-DALDA. CONCLUSIONS: Opiates that activate Mas-related G protein-coupled receptor will degranulate mast cells, activate fibroblasts, and result in intrathecal mass formation. Results suggest a mechanistically rational path forward to safer intrathecal opioid therapeutics.


Subject(s)
Cell Degranulation/drug effects , Fibroblasts/drug effects , Mast Cells/drug effects , Morphine/pharmacology , Receptors, G-Protein-Coupled/physiology , Spine/drug effects , Analgesics, Opioid/administration & dosage , Analgesics, Opioid/pharmacology , Animals , Guinea Pigs , Humans , Infusions, Spinal , Male , Models, Animal , Morphine/administration & dosage , Signal Transduction/physiology
18.
Proc Natl Acad Sci U S A ; 116(16): 7932-7940, 2019 04 16.
Article in English | MEDLINE | ID: mdl-30944217

ABSTRACT

A subset of genes in the human genome are uniquely human and not found in other species. One example is CHRFAM7A, a dominant-negative inhibitor of the antiinflammatory α7 nicotinic acetylcholine receptor (α7nAChR/CHRNA7) that is also a neurotransmitter receptor linked to cognitive function, mental health, and neurodegenerative disease. Here we show that CHRFAM7A blocks ligand binding to both mouse and human α7nAChR, and hypothesized that CHRFAM7A-transgenic mice would allow us to study its biological significance in a tractable animal model of human inflammatory disease, namely SIRS, the systemic inflammatory response syndrome that accompanies severe injury and sepsis. We found that CHRFAM7A increased the hematopoietic stem cell (HSC) reservoir in bone marrow and biased HSC differentiation to the monocyte lineage in vitro. We also observed that while the HSC reservoir was depleted in SIRS, HSCs were spared in CHRFAM7A-transgenic mice and that these mice also had increased immune cell mobilization, myeloid cell differentiation, and a shift to inflammatory monocytes from granulocytes in their inflamed lungs. Together, the findings point to a pathophysiological inflammatory consequence to the emergence of CHRFAM7A in the human genome. To this end, it is interesting to speculate that human genes like CHRFAM7A can account for discrepancies between the effectiveness of drugs like α7nAChR agonists in animal models and human clinical trials for inflammatory and neurodegenerative disease. The findings also support the hypothesis that uniquely human genes may be contributing to underrecognized human-specific differences in resiliency/susceptibility to complications of injury, infection, and inflammation, not to mention the onset of neurodegenerative disease.


Subject(s)
Hematopoietic Stem Cells , alpha7 Nicotinic Acetylcholine Receptor , Animals , Cells, Cultured , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/physiology , Humans , Inflammation/genetics , Inflammation/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , alpha7 Nicotinic Acetylcholine Receptor/genetics , alpha7 Nicotinic Acetylcholine Receptor/immunology , alpha7 Nicotinic Acetylcholine Receptor/metabolism , alpha7 Nicotinic Acetylcholine Receptor/physiology
19.
FASEB J ; 33(5): 6129-6139, 2019 05.
Article in English | MEDLINE | ID: mdl-30715917

ABSTRACT

Healthy repair of cutaneous injury is a coordinated response of inflammatory cells, secreted factors, and biologically active extracellular vesicles (EVs). Although constitutive release of EVs into biologic fluids is a hallmark of cultured cells and tumors, their payload and biologic activity appears to be tightly regulated. We show that Tre-2/Bub2/Cdc16 (TBC1) domain family member 3 (TBC1D3) drives the release of an EV population that causes a decrease in phosphorylation of the transcription factor signal transducer and activator of transcription 3 in naive recipient cells. To explore the biologic activity of EVs in vivo, we used a mouse model of sterile subcutaneous inflammation to determine the payload and biologic activity of EVs released into the microenvironment by committed myeloid lineages and stroma. Expression of TBC1D3 in macrophages altered the payload of their released EVs, including RNA-binding proteins, molecular motors, and proteins regulating secretory pathways. A wound-healing model demonstrated that closure was delayed by EVs released under the control of TBC1D3. We show that modulating the secretory repertoire of a cell regulates EV payload and biologic activity that affects outcomes in tissue repair and establishes a strategy for modifying EVs mediating specific biologic responses.-Qin, S., Dorschner, R. A., Masini, I., Lavoie-Gagne, O., Stahl, P. D., Costantini, T. W., Baird, A., Eliceiri, B. P. TBC1D3 regulates the payload and biological activity of extracellular vesicles that mediate tissue repair.


Subject(s)
Extracellular Vesicles/metabolism , GTPase-Activating Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Wound Healing , Adoptive Transfer , Animals , Extracellular Vesicles/transplantation , GTPase-Activating Proteins/genetics , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Phosphorylation , Proto-Oncogene Proteins/genetics , RAW 264.7 Cells , STAT Transcription Factors/metabolism , Signal Transduction , THP-1 Cells
20.
Adv Biosyst ; 3(10): e1900106, 2019 10.
Article in English | MEDLINE | ID: mdl-32648726

ABSTRACT

Wound healing is a complex process involving diverse changes in multiple cell types where the application of electric fields has been shown to accelerate wound closure. To define the efficacy of therapies based on electric fields, it would be valuable to have a platform to systematically study the effects of electrical stimulation (ES) upon the inflammation phase and the activation of signaling mediators. Here, an in vivo ES model in which flexible electrodes are applied to an animal model for monitoring inflammation in a wound is reported on. Subcutaneous implants of polyvinyl alcohol sponges elicit inflammation response as defined by the infiltration of leukocytes. The wound site is subjected to electric fields using two types of additively fabricated flexible electrode arrays. The sponges are then harvested for flow cytometry analysis to identify changes in the phosphorylation state of intracellular targets. This platform enables studies of molecular mechanisms, as it shows that an application of low-frequency ES ≤0.5 Hz increases phosphorylation of Erk proteins in recruited leukocytes, identifying a signaling pathway that is activated during the healing process.


Subject(s)
Electric Stimulation , Inflammation/immunology , Leukocytes , Phosphorylation/radiation effects , Wound Healing , Animals , Equipment Design , Flow Cytometry/instrumentation , Flow Cytometry/methods , Leukocytes/immunology , Leukocytes/radiation effects , Mice , Mice, Inbred C57BL , Phosphorylation/immunology , Signal Transduction/immunology , Signal Transduction/radiation effects , Wound Healing/immunology , Wound Healing/radiation effects
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