Correlation Between Bacterial Count and Levels of Endotoxin and Inflammatory Mediators During Ex Vivo Treatment of Infection in Human Donor Lungs

AbstractAllogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

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Ex vivo Treatment of Cytomegalovirus in Human Donor Lungs Using a Novel Chemokine-Based Immunotoxin

The Journal of Heart and Lung Transplantation ◽

10.1016/j.healun.2021.10.010 ◽

2021 ◽

Author(s):

Rafaela V.P. Ribeiro ◽

Terrance Ku ◽

Aizhou Wang ◽

Layla Pires ◽

Victor H. Ferreira ◽

...

Keyword(s):

Ex Vivo ◽

Human Donor

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Inflammatory Mediators and Gut Microbial Toxins Drive Colon Tumorigenesis by IL-23 Dependent Mechanism

Cancers ◽

10.3390/cancers13205159 ◽

2021 ◽

Vol 13 (20) ◽

pp. 5159

Author(s):

Janani Panneerselvam ◽

Venkateshwar Madka ◽

Rajani Rai ◽

Katherine T. Morris ◽

Courtney W. Houchen ◽

...

Keyword(s):

Colon Cancer ◽

Dendritic Cells ◽

Tumor Progression ◽

Inflammatory Mediators ◽

Ex Vivo ◽

Colon Tumor ◽

Colonic Tumor ◽

Colon Tumorigenesis ◽

Microbial Toxins

Obesity-associated chronic inflammation predisposes colon cancer risk development. Interleukin-23 (IL-23) is a potential inflammatory mediator linking obesity to chronic colonic inflammation, altered gut microbiome, and colon carcinogenesis. We aimed to elucidate the role of pro-inflammatory eicosanoids and gut bacterial toxins in priming dendritic cells and macrophages for IL-23 secretion to promote colon tumor progression. To investigate the association of IL-23 with obesity and colon tumorigenesis, we utilized TCGA data set and colonic tumors from humans and preclinical models. To understand IL-23 production by inflammatory mediators and gut microbial toxins, we performed several in vitro mechanistic studies to mimic the tumor microenvironment. Colonic tumors were utilized to perform the ex vivo experiments. Our findings showed that IL-23 is elevated in obese individuals, colonic tumors and correlated with reduced disease-free survival. In vitro studies showed that IL-23 treatment increased the colon tumor cell self-renewal, migration, and invasion while disrupting epithelial barrier permeability. Co-culture experiments of educated dendritic cells/macrophages with colon cancer cells significantly increased the tumor aggression by increasing the secretory levels of IL-23, and these observations are further supported by ex vivo rat colonic tumor organotypic experiments. Our results demonstrate gut microbe toxins and eicosanoids facilitate IL-23 production, which plays an important role in obesity-associated colonic tumor progression. This newly identified nexus represents a potential target for the prevention and treatment of obesity-associated colon cancer.

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Abstract 17268: ApoA-I Mimetic Peptide 4F Suppresses the Elevation of Pro-Inflammatory Lipid Mediators in Mouse Models of Inflammatory Bowel Disease

Circulation ◽

10.1161/circ.138.suppl_1.17268 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

David Meriwether ◽

Carmen Volpe ◽

Victor Grijalva ◽

Ellen O’Connor ◽

Nasrin Dorreh ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Mouse Models ◽

Inflammatory Mediators ◽

Bowel Disease ◽

Ex Vivo ◽

Early Stage ◽

Lipid Mediators ◽

Serosal Side ◽

Ussing Chambers ◽

Inflammatory Bowel

Introduction: Inflammatory bowel disease (IBD) has been linked to an increased prevalence of early stage vascular disease. ApoA-I mimetic peptides including 4F are potential therapeutic agents for the treatment of inflammatory diseases including atherosclerosis, and their mechanism of action appears localized to the intestine. We have reported that 4F protects against the development of disease in both the piroxicam-accelerated IL10-/- and myeloid COX2-/- mouse models of IBD. Hypothesis: We previously reported that plasma and lesion levels of oxidized products of linoleic and arachidonic acid correlate with disease in mouse models of atherosclerosis, and that 4F protects against disease in these models while inhibiting accumulation of these pro-inflammatory mediators. We thus sought to determine the complete lipid pro-inflammatory mediator profiles of both the COX2- and IL10-dependent models of IBD, while also determining the effect of 4F on the pro-inflammatory lipid profiles. Methods: We developed and validated a LC-ESI-MS/MS method for determining the levels of 40 lipid inflammatory mediators in both intestinal tissue and plasma, and we analyzed the effects of both disease and 4F upon these mediators in both IBD models. We also employed Ussing chambers to investigate ex vivo the direct effect of 4F on the clearance of pro-inflammatory lipid mediators from intestinal explants and serosal-side lipoproteins. Results: Disease in both models correlated with significantly elevated tissue and plasma levels of multiple lipid pro-inflammatory mediators, while the protective effects of 4F correlated with the significant suppression of most of these mediators. Of interest, 4F inhibited the disease dependent increase of 15HETE, 12HETE, 5HETE, 13HODE, LTB4, 6ketoPGF1α, PGF2α, and TXB2 in the COX2-/- model; and 15HETE, 12HETE, 13HODE, LTB4, and LTE4 in the IL10-/- model. Ex vivo, we showed that 4F could directly clear the pro-inflammatory mediators from inflamed intestinal explants, while also mediating their trans-intestinal efflux from serosal-side lipoproteins. Conclusions: 4F appears to protect against IBD in part by inhibiting the accumulation of pro-inflammatory lipid mediators, through a mechanism that involves the intestinal clearance of these mediators from tissue and plasma.

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Evaluation of Cytotoxicity of Perfluorocarbons for Intraocular Use by Cytotoxicity Test In Vitro in Cell Lines and Human Donor Retina Ex Vivo

Translational Vision Science & Technology ◽

10.1167/tvst.8.5.24 ◽

2019 ◽

Vol 8 (5) ◽

pp. 24 ◽

Cited By ~ 3

Author(s):

Mario R Romano ◽

Mariantonia Ferrara ◽

Claudio Gatto ◽

Barbara Ferrari ◽

Laura Giurgola ◽

...

Keyword(s):

Cell Lines ◽

Ex Vivo ◽

Cytotoxicity Test ◽

Human Donor

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In Vitro and Ex Vivo Evaluation of Nepafenac-Based Cyclodextrin Microparticles for Treatment of Eye Inflammation

Nanomaterials ◽

10.3390/nano10040709 ◽

2020 ◽

Vol 10 (4) ◽

pp. 709 ◽

Cited By ~ 2

Author(s):

Blanca Lorenzo-Veiga ◽

Patricia Diaz-Rodriguez ◽

Carmen Alvarez-Lorenzo ◽

Thorsteinn Loftsson ◽

Hakon Hrafn Sigurdsson

Keyword(s):

Zeta Potential ◽

Physicochemical Properties ◽

Inflammatory Mediators ◽

Ex Vivo ◽

Posterior Segment ◽

Inflammatory Activity ◽

Commercial Formulation ◽

Anti Inflammatory ◽

Negative Zeta Potential

The aim of this study was to design and evaluate novel cyclodextrin (CD)-based aggregate formulations to efficiently deliver nepafenac topically to the eye structure, to treat inflammation and increase nepafenac levels in the posterior segment, thus attenuating the response of inflammatory mediators. The physicochemical properties of nine aggregate formulations containing nepafenac/γ-CD/hydroxypropyl-β (HPβ)-CD complexes as well as their rheological properties, mucoadhesion, ocular irritancy, corneal and scleral permeability, and anti-inflammatory activity were investigated in detail. The results were compared with a commercially available nepafenac suspension, Nevanac® 3 mg/mL. All formulations showed microparticles, neutral pH, and negative zeta potential (–6 to –27 mV). They were non-irritating and nontoxic and showed high permeation through bovine sclera. Formulations containing carboxymethyl cellulose (CMC) showed greater anti-inflammatory activity, even higher than the commercial formulation, Nevanac® 0.3%. The optimized formulations represent an opportunity for topical instillation of drugs to the posterior segment of the eye.

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Insights Into Thrombopoiesis From Infused Human Megakaryocytes Into Mice

Blood ◽

10.1182/blood.v122.21.1165.1165 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1165-1165

Author(s):

Yuhuan Wang ◽

Vincent M. Hayes ◽

Prasuna Paluru ◽

Stella T. Chou ◽

Deborah L. French ◽

...

Keyword(s):

Half Life ◽

Size Range ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Human Donor ◽

Nsg Mice ◽

Infusion Study ◽

Recipient Animal ◽

Post Infusion

Abstract Thrombopoiesis is the process by which megakaryocytes (Megs) release platelets (Plts), but issues remain as to the detailed in vivo mechanisms underlying this process. We now report new insights into this process by studying infused human Megs into immunocompromized NOD/SCID, gamma-interferon deleted (NSG) mice. Prior in situ microscopy has suggested that Megs release varied-size cytoplasmic fragments up to whole Megs in size into the medullary vascular space. Other studies have suggested that at least a portion of thrombopoiesis occurs by Megs lodged in the lungs. We previously infused ex vivo-generated murine Megs into mice and found that these Megs become entrapped in the animals’ lungs, and in <1.5 hrs, release functional Plts (termed here “Meg-Plts”) that have a similar half-life as infused mouse donor-derived Plts (termed here “Donor-Plts”). To better understand the biology of thrombopoiesis, we have infused ex vivo-generated human Megs into NSG mice. These studies replicated many of the observations seen with infused murine Megs: Human Megs were entrapped in the lungs with delayed release of human Meg-Plts, and these Meg-Plts had the same half-life as infused human Donor-Plts. Human Plts differ from murine Plts in size so this parameter was analyzed following infusion of human Megs using forward cell scatter analysis. We noted that 10 mins post-infusion, the Meg-Plt size range was wide and displayed a non-bell-shaped distribution. This distribution was in contrast to the tight bell-shaped curves seen for the endogenous murine Plts and for infused human Donor-Plts. However, by 3 hrs post-human Meg infusion - at the time of peak Meg-Plt counts - the human Meg-Plts now displayed an identical bell-shaped distribution curve as infused human Donor-Plt. The smaller, human Meg-Plts had disappeared. The size and distribution of these Meg-Plts then remained near identical to Donor-Plts for the remaining portion of the 48 hr post-infusion study. However, after impairing macrophage clearance in NSG recipient mice with clodronate-ladened liposome infusion, the small Meg-Plts did not disappear and were present at 48 hrs. Using thiazole orange (TO) to stain platelets for RNA content, we noted that ∼70% of all Meg-Plts were initially TO+ compared to the steady-state of ∼10% for mouse endogenous platelets. This high TO+ state decreased to near 10% by 24 hrs post-infusion. Up to ∼6 hrs, all of the large Meg-Plts were TO+, while the smaller-sized Meg-Plts were predominantly TO-. Unless the mice were treated with clodronate-ladened liposomes, these TO-, small Meg-Plts disappeared before 6 hrs. In conclusion, these data support that ex vivo-generated human Megs release physiologic platelets in the pulmonary vascular bed of NSG mice with the same size range/distribution and survival as infused human Donor-Plts. Mean Meg-Plt size depends on the species of origin of the infused Megs rather than on the species of the recipient animal. We did not detect large Meg cytoplasmic fragments that underwent further size reduction although our technique may not be capable of detecting small numbers of such fragments or the small size changes that would accompany platelet maturation from preplatelets. Our data also suggest that Megs generated in culture release a wide size range of non-physiologic Plt-like particles that when infused are cleared rapidly by macrophages. Disclosures: No relevant conflicts of interest to declare.

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High CXCL6 drives matrix expression and correlate with markers of poor outcome in IPF

10.1101/2021.06.22.449424 ◽

2021 ◽

Author(s):

Harinath Bahudhanapati ◽

Jiangning Tan ◽

Rosa-Marie Apel ◽

Benjamin Seeliger ◽

Xiaoyun Li ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Ex Vivo ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Collagen I ◽

Lung Fibroblasts ◽

Dependent Manner ◽

Human Donor ◽

Poor Outcomes ◽

Matrix Expression

Signaling via G protein-coupled receptors (GPCRs) can modulate levels of cyclic adenosine monophosphate (cAMP) and shape the functions of fibroblasts in idiopathic pulmonary fibrosis (IPF). We have identified Chemokine (C-X-C) Motif Ligand 6 (CXCL6) as a potential pro-fibrotic GPCR ligand. We tested the function of CXCL6 in ex vivo human donor and fibrotic lung fibroblasts and in an animal model of pulmonary fibrosis. We also measured levels of CXCL6 in the blood and bronchoalveolar lavage (BAL) of patients with IPF. CXCL6 decreased cAMP levels in a dose-dependent manner in Donor and IPF Fibroblasts. CXCL6 mRNA and protein were localized to epithelial cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased Collagen I and α-SMA levels in Donor and IPF Fibroblasts. Silencing of CXCR1/2 as well as Reparixin, a CXCR1/2 inhibitor, blocked effects of CXCL6. Treprostinil blocked effects of CXCL6 only on levels of α-SMA but not on Collagen I. CXCL6 levels in the BAL of two separate cohorts of patients with IPF was associated with poor survival. We conclude that high CXCL6 drives fibroblast function and correlates with poor outcomes in IPF.

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