scholarly journals Investigating the Early Events after Skin-Barrier Disruption Using Microdialysis—A Human Ex Vivo Skin Model

Dermato ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 47-58
Author(s):  
Katrine Baumann ◽  
Niels Peter Hell Knudsen ◽  
Anne-Sofie Østergaard Gadsbøll ◽  
Anders Woetmann ◽  
Per Stahl Skov
Keyword(s):  
Interstitial Fluid ◽  
Ex Vivo ◽  
Skin Barrier ◽  
Microdialysis Sampling ◽  
Sample Analysis ◽  
Skin Model ◽  
Barrier Disruption ◽  
Gm Csf ◽  
Tnf Α ◽  
Subsequent Sample

Skin-barrier restoration following abrasive trauma is facilitated by mediator release from skin-resident cells, a process that has been investigated primarily in mice or simplified human systems with previous studies focusing on a limited number of biomarkers. Here, we demonstrate how early events caused by skin-barrier disruption can be studied in a human ex vivo skin model. Ten relevant biomarkers were recovered from the interstitial fluid by skin microdialysis with subsequent sample analysis using a multiplex platform. As a control, the biomarker profiles obtained from microdialysis sampling were compared to profiles of skin biopsy homogenates. We found that nine (GM-CSF, CXCL1/GROα, CXCL8/IL-8 CXCL10/IP-10, IL-1α, IL-6, MIF, TNF-α, and VEGF) of the 10 biomarkers were significantly upregulated in response to abrasive trauma. Only dialysate levels of CCL27/CTACK were unaffected by skin abrasion. Biomarker levels in the homogenates corresponded to dialysate levels for CCL27/CTACK, CXCL1/GROα, CXCL8/IL-8, and IL-6. However, IL-1α showed an inverse trend in response to trauma, and biopsy levels of MIF were unchanged. GM-CSF, CXCL10/IP-10, TNF-α, and VEGF were not detected in the biopsy homogenates. Our results suggest that the human ex vivo skin model is a reliable approach to study early events after disruption of the skin barrier.

Interterleukin-6 (IL-6) Produced by Monocyte Activation Reduces Dendritic Cell (DC) Differentiation in Patients with Multiple Myeloma (MM) and Malignant Lymphoma (ML): Role of CNTO 328, an Anti-IL-6 Monoclonal Antibody (Mab) and Possible Clinical Applications.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2662-2662
Author(s):  
Jean-Francois Rossi ◽  
Anne-Marie Conge ◽  
Catherine Barjot ◽  
Mohamed H. Zaki ◽  
Marian T. Nakada ◽  
...  
Keyword(s):  
Large Scale ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Human Albumin ◽  
Autologous Tumor ◽  
Vaccination Programs ◽  
Gm Csf ◽  
Free Process ◽  
Tnf Α ◽  
Dc Maturation

Abstract We developed a serum-free process in a closed system using culture cassettes and bags for large-scale and clinical-grade DC vaccination, accepted by the “Afssaps-French drug Agency” (Tarte K. et al. Leukemia2000; 14:2152 & patent). Intermediate mature DCs are generated from mononucleated cells obtained by mobilized leukapheresis, followed by Mo selection using adherence in specific cassettes (CLINIcell, Mabiol). Non-adherent cells are removed and Mo are cultured for 5 days (D) in X-VIVO15 medium (Cambrex) with 2% of human albumin, 100ng/ml of GM-CSF (Leukine, Berlex) and 25 ng/ml of IL-4 (CellGenix-Cellgen). At D5, immature DCs are harvested, pulsed with autologous tumor lysate (or peptides) for 4 h in X-VIVO15 medium + GM-CSF (100ng/ml) and maturation factors (TNF-α: 20ng/ml, CellGenix-CellGen, and PGE2: 100ng/ml; Prostine, Pharmacia). Maturation of DCs was allowed to proceed for 20 h with TNF-α and PGE2. Mo-conditioned media, or IL-6 as well as IL-1 are used for enhancing ex vivo DC maturation by different groups in spite of the fact that IL-6 has been described as a blocker of DC differentiation from CD34+ cells particularly in MM. We demonstrated that in our process, IL-6 is produced by activated Mo during their selection (mean= 378pg/mL, range 37–1219). The amount of the IL-6 released in the medium correlated with the % of CD14+ cells obtained at D5 (CD14<2.8%: mean IL-6=73.1 pg/mL; CD14>22.6%: mean IL-6=682.9 pg/mL), indicating that the intrinsic production of IL-6 is one major parameter of variability of the cellular product. By adding IL-6 from D1 to D5, the percentage of CD14+ cells at D5 was enhanced by a mean of 23-fold in samples from patients with MM (n=7) and 17-fold in ML (n=7). The modifications of other DCs markers including CD1a, CD 84 and CCR7 were modest. By using CNTO 328, an anti-IL-6 MAb (Centocor Inc) at 1–10μg/mL, we totally blocked the activity of added IL-6 and samples with high IL-6 intrinsic production, with a reduction of CD14+ cells at D5. In contrast, neither IL-6 nor CNTO 328 had any activity on terminal DC maturation after D5. IL-6 and CNTO328 are tested on DC functions. This means that in B-cell malignancies and other solid tumors with high levels of circulating IL-6: 1) anti-IL-6 treatment such as CNTO 328 may be associated with active immune therapy, including vaccinations; 2) mature and intermediate mature DCs are the only cells to be administered in vaccination programs because of a de-differentiation effect of immature DCs due to IL-6; 3) anti-IL-6 MAbs, particularly CNTO 328 could be added for ex vivo DC differentiation, instead of IL-6. mean % (range) of CD14+ cells at Day5 samples MM ML Control 2.9 (0.1–7.1) 12.2 (0–44.8) IL-6 (100ng/mL) 20 (6–35) 34.2 (0–71.4) IL-6+CNTO328 1μg/mL 2.8 (0.5–7.5) 15.7 (0–45.6) IL-6+CNTO328 10μg/mL 0.4 (0–0.8) 6.8 (0–20.3) CNTO328 1μg/mL 0.4 (0.1–0.7) 6.5 (0–19.5) CNTO328 10μg/mL 0.2 (0–0.4) 5.3 (0–15.3)

scholarly journals Ex Vivo Live Full-Thickness Porcine Skin Model as a Versatile In Vitro Testing Method for Skin Barrier Research

2021 ◽  
Vol 22 (2) ◽  
pp. 657
Author(s):  
Jee-Hyun Hwang ◽  
Haengdueng Jeong ◽  
Nahyun Lee ◽  
Sumin Hur ◽  
Nakyum Lee ◽  
...  
Keyword(s):  
Barrier Function ◽  
Ex Vivo ◽  
Animal Experiments ◽  
Skin Barrier ◽  
Skin Barrier Function ◽  
Protective Effects ◽  
Skin Model ◽  
Porcine Skin ◽  
Single Exposure ◽  
Cosmetic Ingredients

Since the European Union (EU) announced their animal testing ban in 2013, all animal experiments related to cosmetics have been prohibited, creating a demand for alternatives to animal experiments for skin studies. Here, we investigated whether an ex vivo live porcine skin model can be employed to study the safety and skin barrier-improving effects of hydroxyacids widely used in cosmetics for keratolytic peels. Glycolic acid (1–10%), salicylic acid (0.2–2%), and lactobionic acid (1.2–12%) were used as representative substances for α-hydroxyacid (AHA), β-hydroxyacid (BHA), and polyhydroxyacid (PHA), respectively. When hydroxyacids were applied at high concentrations on the porcine skin every other day for 6 days, tissue viability was reduced to 50–80%, suggesting that the toxicity of cosmetic ingredients can be evaluated with this model. Based on tissue viability, the treatment scheme was changed to a single exposure for 20 min. The protective effects of a single exposure of hydroxyacids on skin barrier function were evaluated by examining rhodamine permeability and epidermal structural components of barrier function using immunohistochemistry (IHC) and immunofluorescence (IF) staining. Lactobionic acid (PHAs) improved skin barrier function most compared to other AHAs and BHAs. Most importantly, trans-epidermal water loss (TEWL), an important functional marker of skin barrier function, could be measured with this model, which confirmed the significant skin barrier-protective effects of PHAs. Collectively, we demonstrated that the ex vivo live full-thickness porcine skin model can be an excellent alternative to animal experiments for skin studies on the safety and efficacy of cosmetic ingredients.

scholarly journals Defining the inflammatory signature of human lung explant tissue in the presence and absence of glucocorticoid

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 460 ◽  
Author(s):  
Tracy L Rimington ◽  
Emily Hodge ◽  
Charlotte K Billington ◽  
Sangita Bhaker ◽  
Binaya K C ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Human Lung ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Chronic Obstructive ◽  
Interleukin 1 Beta ◽  
Gm Csf ◽  
Tnf Α ◽  
Anti Inflammatory

Background: Airway inflammation is a feature of many respiratory diseases and there is a need for newer, more effective anti-inflammatory compounds. The aim of this study was to develop an ex vivo human lung explant model which can be used to help study the mechanisms underlying inflammatory responses and which can provide a tool to aid drug discovery for inflammatory respiratory diseases such as asthma and COPD. Method: Parenchymal lung tissue from 6 individual donors was dissected and cultured with two pro-inflammatory stimuli, lipopolysaccharide (LPS) (1 µg/ml) and interleukin-1 beta (IL-1β) (10 ng/ml) in the presence or absence of dexamethasone (1 µM).  Inflammatory responses were assessed using Luminex analysis of tissue culture supernatants to measure levels of 21 chemokines, growth factors and cytokines. Results: A robust and reproducible inflammatory signal was detected across all donors for 12 of the analytes measured following LPS stimulation with a modest fold increase (<2-fold) in levels of CCL22, IL-4, and IL-2; increases of 2-4-fold in levels of CXCL8, VEGF and IL-6 and increases >4-fold in CCL3, CCL4, GM-CSF, IL-10, TNF-α and IL-1β.  The inflammatory signal induced by IL-1β stimulation was less than that observed with LPS but resulted in elevated levels of 7 analytes (CXCL8, CCL3, CCL4, GM-CSF, IL-6, IL-10 and TNF-α).  The inflammatory responses induced by both stimulations was supressed by dexamethasone for the majority of analytes. Conclusions: These data provide proof of concept that this ex vivo human lung explant model is responsive to inflammatory signals and could be used to investigate the anti-inflammatory effects of existing and novel compounds.  In addition this model could be used to help define the mechanisms and pathways involved in development of inflammatory airway disease. Abbreviations: COPD: Chronic Obstructive Pulmonary Disease; ICS: inhaled corticosteroids; LPS: lipopolysaccharide; IL-1β: interleukin-1 beta; PSF: penicillin, streptomycin and fungizone

scholarly journals Physiological and Molecular Effects of in vivo and ex vivo Mild Skin Barrier Disruption

2018 ◽  
Vol 31 (3) ◽  
pp. 115-124 ◽  
Author(s):  
Eva K.B. Pfannes ◽  
Lina Weiss ◽  
Sabrina Hadam ◽  
Jessica Gonnet ◽  
Béhazine Combardière ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Skin Barrier ◽  
Barrier Disruption ◽  
Molecular Effects

scholarly journals Ex Vivo Cytokine Release, Determined by a Multiplex Cytokine Assay, in Response to Coccidioidal Antigen Stimulation of Whole Blood among Subjects with Recently Diagnosed Primary Pulmonary Coccidioidomycosis

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Neil M. Ampel ◽  
Ian Robey ◽  
Chinh T. Nguyen ◽  
Brentin Roller ◽  
Jessica August ◽  
...  
Keyword(s):  
Antifungal Therapy ◽  
Whole Blood ◽  
Ex Vivo ◽  
Valley Fever ◽  
Blood Samples ◽  
Antigen Stimulation ◽  
Gm Csf ◽  
Tnf Α ◽  
Inflammatory Proteins ◽  
Ifn Γ

ABSTRACT The elements of the cellular immune response in human coccidioidomycosis remain undefined. We examined the ex vivo release of an array of inflammatory proteins in response to incubation with a coccidioidal antigen preparation to ascertain which of these might be associated with diagnosis and outcome. Patients with a recent diagnosis of primary pulmonary coccidioidomycosis and a control group of healthy subjects were studied. Blood samples were incubated for 18 h with T27K, a soluble coccidioidal preparation containing multiple glycosylated antigens, and the supernatant was assayed for inflammatory proteins using the multiplex Luminex system. The presentation and course of illness were compared to the levels of the inflammatory proteins. Among the 31 subjects studied, the median time from diagnosis to assay was 15 days. Of the 30 inflammatory proteins measured, the levels of only 7 proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor alpha (IL-1RA), interleukin-1β (IL-1β), interferon gamma (IFN-γ), IL-2, IL-13, and tumor necrosis factor alpha (TNF-α), were more than 10-fold above the levels seen without antigen stimulation. The levels of IFN-γ and IL-2 were significantly elevated in those subjects not receiving triazole antifungal therapy compared to those who were receiving triazole antifungal therapy. While the levels of IL-1RA were nonspecifically elevated, elevated levels of IL-13 were seen only in those with active pulmonary coccidioidomycosis. Only six cytokines were specifically increased in subjects with recently diagnosed primary pulmonary coccidioidomycosis. While IFN-γ, IL-2, and TNF-α have been previously noted, the finding of elevated levels of the innate cytokines GM-CSF and IL-1β could suggest that these, as well as IL-13, are early and specific markers for pulmonary coccidioidomycosis. IMPORTANCE Coccidioidomycosis, commonly known as Valley fever, is a common pneumonia in the southwestern United States. In this paper, we examined the release of 30 inflammatory proteins in whole-blood samples obtained from persons with coccidioidal pneumonia after the blood samples were incubated with a preparation made from the causative fungus, Coccidioides . We found that six of these proteins, all cytokines, were specifically released in high concentrations in these patients. Three of the cytokines were seen very early in disease, and an assay for all six might serve as a marker for the early diagnosis of Valley fever.

scholarly journals Skin barrier disruption by acetone: observations in a hairless mouse skin model

2009 ◽  
Vol 301 (8) ◽  
pp. 609-613 ◽  
Author(s):  
Robert Rissmann ◽  
Marion H. M. Oudshoorn ◽  
Wim E. Hennink ◽  
Maria Ponec ◽  
Joke A. Bouwstra
Keyword(s):  
Mouse Skin ◽  
Skin Barrier ◽  
Hairless Mouse ◽  
Skin Model ◽  
Hairless Mouse Skin ◽  
Barrier Disruption

scholarly journals Serine Protease-Mediated Cutaneous Inflammation: Characterization of an Ex Vivo Skin Model for the Assessment of Dexamethasone-Loaded Core Multishell-Nanocarriers

Pharmaceutics ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 862
Author(s):  
Janna Frombach ◽  
Fiorenza Rancan ◽  
Katharina Kübrich ◽  
Fabian Schumacher ◽  
Michael Unbehauen ◽  
...  
Keyword(s):  
Serine Protease ◽  
Ex Vivo ◽  
Biological Effects ◽  
Skin Barrier ◽  
Fold Increase ◽  
Transepidermal Water Loss ◽  
Moderate Increase ◽  
Skin Model ◽  
Proinflammatory Mediators ◽  
Anti Inflammatory

Standard experimental set-ups for the assessment of skin penetration are typically performed on skin explants with an intact skin barrier or after a partial mechanical or chemical perturbation of the stratum corneum, but they do not take into account biochemical changes. Among the various pathological alterations in inflamed skin, aberrant serine protease (SP) activity directly affects the biochemical environment in the superficial compartments, which interact with topically applied formulations. It further impacts the skin barrier structure and is a key regulator of inflammatory mediators. Herein, we used short-term cultures of ex vivo human skin treated with trypsin and plasmin as inflammatory stimuli to assess the penetration and biological effects of the anti-inflammatory drug dexamethasone (DXM), encapsulated in core multishell-nanocarriers (CMS-NC), when compared to a standard cream formulation. Despite a high interindividual variability, the combined pretreatment of the skin resulted in an average 2.5-fold increase of the transepidermal water loss and swelling of the epidermis, as assessed by optical coherence tomography, as well as in a moderate increase of a broad spectrum of proinflammatory mediators of clinical relevance. The topical application of DXM-loaded CMS-NC or DXM standard cream revealed an increased penetration into SP-treated skin when compared to untreated control skin with an intact barrier. Both formulations, however, delivered sufficient amounts of DXM to effectively suppress the production of interleukin-6 (IL-6), interleukin-8 (IL-8) and Thymic Stromal Lymphopoietin (TSLP). In conclusion, we suggest that the herein presented ex vivo inflammatory skin model is functional and could improve the selection of promising drug delivery strategies for anti-inflammatory compounds at early stages of development.

scholarly journals Human neutrophils depend on extrinsic factors produced by monocytes for their survival response to TLR4 stimulation

2019 ◽  
Vol 25 (8) ◽  
pp. 473-486
Author(s):  
Shuvasree SenGupta ◽  
Madhavi J Rane ◽  
Silvia M Uriarte ◽  
Cassandra Woolley ◽  
Thomas C Mitchell
Keyword(s):  
Molecular Mechanisms ◽  
Lipid A ◽  
Ex Vivo ◽  
Cell Types ◽  
Human Neutrophils ◽  
Direct Stimulation ◽  
Extrinsic Factors ◽  
Gm Csf ◽  
Tnf Α ◽  
Neutrophil Survival

LPS delays neutrophil apoptosis by a process generally assumed to involve cell-intrinsic TLR4 signaling. However, neutrophil survival responses to LPS have been reported to be monocyte-dependent, which would indicate more complexity than is currently appreciated. We compared the survival responses of conventionally purified vs highly purified neutrophils to confirm or refute the need for secondary cell-types and to identify the cellular or molecular mechanisms involved. Direct stimulation of TLR4 failed to extend the survival of highly purified neutrophils, but survival activity was retained in less pure neutrophil preparations containing low numbers of eosinophils, monocytes, platelets and CD3+ lymphocytes. Sequential depletions identified monocytes as the only cell type required. Transfer of culture supernatants after lipid A-conditioning revealed that purified monocytes were sufficient for production of nearly all of the survival activity observed in mixed populations. The survival factors secreted upon TLR4 stimulation remain unidentified, but were not correlated with IL-1β, IL-6 or TNF-α nor could survival activity be inhibited by Ab blockade of IL-8 or of several other candidate factors other than endogenously produced GM-CSF, which was responsible for about one-tenth of the survival activity present in conditioned supernatants. These observations confirm that ex vivo neutrophil survival responses to TLR4 agonists are not cell intrinsic and involve potentially novel factors secreted by TLR4-stimulated monocytes.

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