scholarly journals Immunohistochemical analysis of the inflammatory reaction within the implantation bed of a collagen hemostatic biomaterial

2021 ◽  
Author(s):  
◽  
Carlos Arturo Herrera Vizcaino
Keyword(s):  
Wound Healing ◽  
Inflammatory Response ◽  
Inflammatory Cytokines ◽  
Degradation Rate ◽  
Ex Vivo ◽  
The Body ◽  
Cellular Reaction ◽  
Intergroup Comparison ◽  
Pro Inflammatory Cytokines

Current research on medical biomaterials have shown that the physical and chemical characteristics of biomaterials determine the body inflammatory cellular reaction after their implantation. The aim of this study was to evaluate the individual effects of the physical characteristics over the initial biomaterial-cellular interaction and the inflammatory cellular reaction. For this purpose, an equine-derived collagen hemostatic sponge (E-CHS) was modified by pressing and evaluated using ex vivo, in vitro and in vivo methods. The E-CHS was pressed by applying constant pressure (6.47± 0.85 N) for 2 min using a sterile stainless-steel cylinder and cut in segments of 1cm2. Subsequently, E-CHS and the pressed equine-derived collagen hemostatic sponge (P-E-CHS) were studied as two independent biomaterials and compared to a control group (CG). A blood concentrate containing inflammatory cells known as platelet rich fibrin (PRF) was used to mimic the initial biomaterial-cell interaction and to measure the absorption coefficient of the biomaterials to liquid PRF (iPAC). Additionally, the biomaterials were cultivated together with PRF for 3 and 6 days to measure the induction of pro-inflammatory cytokines (TNF-α and IL-8). The results were obtained through enzyme-linked immunosorbent assay (ELISA) and histological methods. PRF cultivated without biomaterials served as the CG. Additionally, the biomaterials were evaluated in vivo using a subcutaneous model in Wistar rats and compared to sham operated animals (CG) representing physiologic wound healing. After 3, 15 and 30 days, the explanted samples were evaluated using histochemical and immunohistochemical (IHC) staining using the following markers: CD68 (pan macrophages), CCR7 (pro-inflammatory macrophages, M1), CD206 (pro-wound healing macrophages, M2) and α-Smooth Muscle Actin (α-SMA; vessel identification). After the mixture of liquid PRF with both biomaterials for 15 minutes, the ex vivo results showed that E-CHS was penetrated by cells, whereas P-E-CHS was cell-occlusive. Additionally, P-E-CHS induced a higher release of pro-inflammatory cytokines compared to liquid PRF alone (CG) and E-CHS after 3 days (P< 0.05). Although the biomaterial was pressed, the difference of the iPAC value did not show statistical differences. In vivo, the CG induced at day 3 a higher inflammatory response compared to the experimental groups (EG) (P< 0.05). The intergroup comparison showed that P-E-CHS induced a higher presence of macrophages (CD68+/CC7+) compared to E-CHS at day 3 (P< 0.05). Only CD68+/CCR7+ mononuclear cells (MNCs) were observed without multinucleated giant cells (MNGCs). After 15 days, the presence of macrophages (CD68+ P<0.01 /CCR7+ P<0.001 /CD206+ P<0.05) reduced considerably in the CG. On the contrary, the inflammatory response increased in the EGs (CD68+/CCR7+). The intergroup comparison showed that this increment was statistically significant when comparing E-CHS and P-E-CHS to the CG at day 15 (P<0.01 and P< 0.05 respectively). At this time point, a reduced number of MNGCs were observed in the EGs. In the CG no MNGCs were observed. Furthermore, E-CHS showed a faster degradation rate and was fully invaded by cells and vessels formed in its interior region. On the other hand, P-E-CHS remained occlusive to cell penetration and vessels were formed only in the periphery. After 30 days, the cellular reaction shifted to a higher number of M2 macrophages (CD260+) in all groups and a reduced presence of CD68+ and CCR7+ MNCs. Both biomaterials degraded and only small fragments were found in the implantation bed surrounded by MNGCs (CCR7+). These results are of high clinical relevance and show that changes in biomaterial properties have a significant impact on their interaction with the body. They also serve as insight into the possibility to develop versatile biomaterials with different applications. For example, E-CHs can be applied to support hemostasis in a bleeding alveolar socket and P-E-CHs by being cell occlusive and having a delayed degradation rate can be applied for guided bone and tissue regeneration.

scholarly journals A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroto Nakajima ◽  
Atsushi Miyashita ◽  
Hiroshi Hamamoto ◽  
Kazuhisa Sekimizu
Keyword(s):  
Inflammatory Cytokines ◽  
Immune Cells ◽  
Ex Vivo ◽  
Large Bubble ◽  
Suitable Model ◽  
Bacterial Cells ◽  
Functional Impairments ◽  
Gene Expressions ◽  
Pro Inflammatory Cytokines

AbstractIn this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

scholarly journals A Novel Application of Bubble-eye Strain of Carassius Auratus for Ex Vivo Fish Immunological Studies

2021 ◽  
Author(s):  
Hiroto Nakajima ◽  
Atsushi Miyashita ◽  
Hiroshi Hamamoto ◽  
Kazuhisa Sekimizu
Keyword(s):  
Inflammatory Cytokines ◽  
Immune Cells ◽  
Ex Vivo ◽  
Large Bubble ◽  
Suitable Model ◽  
Bacterial Cells ◽  
Functional Impairments ◽  
Gene Expressions ◽  
Pro Inflammatory Cytokines

Abstract In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at high temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and high rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

scholarly journals Modification of collagen-based sponges can induce an upshift of the early inflammatory response and a chronic inflammatory reaction led by M1 macrophages: an in vivo study

2020 ◽  
Vol 24 (10) ◽  
pp. 3485-3500 ◽  
Author(s):  
C. Herrera-Vizcaíno ◽  
S. Al-Maawi ◽  
R. Sader ◽  
C. J. Kirkpatrick ◽  
J. Choukroun ◽  
...  
Keyword(s):  
Wound Healing ◽  
Inflammatory Response ◽  
Inflammatory Reaction ◽  
Ex Vivo ◽  
Cell Interaction ◽  
Control Group ◽  
Ex Vivo Model ◽  
Inflammatory Proteins ◽  
Time Point

Abstract Background The present study evaluated the cellular tissue reaction of two equine-derived collagen hemostatic sponges (E-CHS), which differed in thickness after pressing, over 30 days in vivo. The inflammatory response during physiological wound healing in sham-operated animals was used as control group. Material and methods First, the E-CHS was pressed by applying constant pressure (6.47 ± 0.85 N) for 2 min using a sterile stainless-steel cylinder until the material was uniformly flattened. Consequently, the original (E-CHS), the pressed (P-E-CHS), as well as the control group (CG; sham operation) were studied independently. The 3 groups were evaluated in vivo after subcutaneous implantation in Wistar rats during 3, 15, and 30 days. Histochemical and immunohistochemical methods provided observations of biomaterial degradation rate, cellular inflammatory response, and vascularization pattern. A derivative of human blood known as platelet-rich fibrin (PRF) was used as an ex vivo model to simulate the initial biomaterial-cell interaction. Segments of E-CHS and P-E-CHS were cultivated for 3 and 6 days with PRF, and the release of pro-inflammatory proteins was measured using ELISA. PRF cultivated alone was used as a control group. Results At day 3, the CG induced a statistically significant higher presence of monocytes/macrophages (CD68+), pro-inflammatory macrophages (M1; CCR7+), and pro-wound healing macrophages (M2; CD206+) compared to E-CHS and P-E-CHS. At the same time point, P-E-CHS induced a statistically significant higher presence of CD68+ cells compared to E-CHS. After 15 days, E-CHS was invaded by cells and vessels and showed a faster disintegration rate compared to P-E-CHS. On the contrary, cells and vessels were located only in the outer region of P-E-CHS and the biomaterial did not lose its structure and accordingly did not undergo disintegration. The experimental groups induced similar inflammatory reaction primarily with positive pro-inflammatory CD68+/CCR7+ macrophages and a low presence of multinucleated giant cells (MNGCs). At this time point, significantly lower CD68+/CCR7+ macrophages and no MNGCs were detected within the CG when compared to the experimental groups (P < 0.05). After 30 days, E-CHS and P-E-CHS were fully degraded. All groups showed similar inflammatory reaction shifted to a higher presence CD206+ macrophages. A low number of CCR7+ MNGCs were still observable in the implantation bed of both experimental groups. In the ex vivo model, the cells and fibrin from PRF penetrated E-CHS. However, in the case of P-E-CHS, the cells and fibrin stayed on the surface and did not penetrate towards materials central regions. The cultivation of P-E-CHS with PRF induced a statically significant higher release of pro-inflammatory proteins compared to the CG and E-CHS after 3 days. Conclusion Altering the original presentation of a hemostatic sponge biomaterial by pressing modified the initial biomaterial-cell interaction, delayed the early biomaterial’s degradation rate, and altered the vascularization pattern. A pressed biomaterial seems to induce a higher inflammatory reaction at early time points. However, altering the biomaterial did not modify the polarization pattern of macrophages compared to physiologic wound healing. The ex vivo model using PRF was shown to be an effective model to simulate the initial biomaterial-cell interaction in vivo. Clinical relevance A pressed hemostatic sponge could be applied for guided tissue regeneration and guided bone regeneration. In that sense, within the limitations of this study, the results show that the same biomaterial may have two specific clinical indications.

scholarly journals IMMU-07. TUMOR STROMA–TARGETING ANTIBODY-CYTOKINE CONJUGATES TO CONVERT THE IMMUNOLOGICALLY COLD GLIOMA MICROENVIRONMENT INTO A HOT ONE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii106-ii106
Author(s):  
Tobias Weiss ◽  
Emanuele Puca ◽  
Manuela Silginer ◽  
Teresa Hemmerle ◽  
Shila Pazahr ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Targeted Delivery ◽  
Fusion Proteins ◽  
Tumor Necrosis ◽  
Ex Vivo ◽  
Systemic Administration ◽  
Target Antigen ◽  
Tumor Site ◽  
Pro Inflammatory Cytokines

Abstract Glioblastoma is an immunological “desert”. The administration of pro-inflammatory cytokines could shift the balance between tumor-associated immune suppression and anti-tumor immunity but the systemic administration of therapeutically active doses of pro-inflammatory cytokines is hampered by toxic side effects. We investigated different antibody-cytokine fusion products that enable a targeted delivery of interleukin (IL-) 2, IL-12 or tumor-necrosis factor (TNF)α to the tumor site upon systemic administration by binding to a tumor-specific epitope of fibronectin. We confirmed the target antigen expression in ex vivo sections of orthotopic syngeneic glioma mouse models and human glioblastoma samples and characterized the distribution of these antibody-conjugates in glioma-bearing mice upon systemic administration using in vivo imaging. Subsequently, we demonstrated potent anti-tumor activity of these antibody-fusion proteins in fully immunocompetent orthotopic mouse glioma models and characterized their mode of action. We also translated this immunotherapeutic strategy to treat patients with recurrent glioblastoma systemically with an antibody-fusion protein that enables the targeted delivery of TNFα to the tumor site. This was well tolerated, led to a treatment-associated tumor necrosis and increased the number of tumor-infiltrating T cells. This work builds a basis for future studies with antibody-cytokine fusion proteins as a promising treatment strategy for central nervous system tumors.

scholarly journals Unmethylated CpG motif-containing genomic DNA fragment of Bacillus calmette-guerin promotes macrophage functions through TLR9-mediated activation of NF-κB and MAPKs signaling pathways

2019 ◽  
Vol 26 (3) ◽  
pp. 183-203 ◽  
Author(s):  
Junli Li ◽  
Lili Fu ◽  
Guozhi Wang ◽  
Selvakumar Subbian ◽  
Chuan Qin ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Immune Cell ◽  
Ex Vivo ◽  
Effector Functions ◽  
Cell Functions ◽  
Cpg Motif ◽  
Pro Inflammatory Cytokines

The potency of synthetic CpG-oligo-deoxynucleotides (CpG-ODNs) adjuvants in modulating the immune cell functions through the TLR pathway has been tested and reported previously. However, the cellular signaling involved in the stimulation of macrophages by natural, CpG motif-containing adjuvant and the effector functions modulated by such stimulation has not been well studied. Here, we used in vitro and ex vivo murine macrophage assay systems, and mouse model of in vivo stimulation to explore the signaling pathway and the effector functions mediated by BC01. Results show that BC01 can induce the production of TNF-α and MCP-1 in macrophages by up-regulating the activation of NF-κB and MAPKs signaling pathway, and elevated the expression of MHC-II, CD40, CD80, and CD86. Upon stimulation with BC01, the peritoneal macrophages isolated from TLR9−/− mice produced significantly low levels of pro-inflammatory cytokines, attenuated the activation of NF-κB and MAPKs signaling pathways, and showed reduced phagocytosis. Following in vivo stimulation with BC01, the TLR9−/− mice produced significantly lower levels of pro-inflammatory cytokines in the serum and lymph nodes showed reduced cell proliferation. These results indicate that BC01 is an efficient agonist of TLR9 that can significantly enhance the host-protective immune functions of macrophages.

scholarly journals Role of cholinergic anti-inflammatory pathway in protecting sepsis-induced acute lung injury through regulation of the dendritic cells

2021 ◽  
Author(s):  
Ruiting Li ◽  
Xuemei Hu ◽  
Huibin Chen ◽  
Yin Yuan ◽  
Huiling Guo ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Response ◽  
Inflammatory Cytokines ◽  
Mice Model ◽  
Pro Inflammatory Cytokines

Abstract Background The cholinergic anti-inflammatory pathway (CAP) connects the immune response system and the nervous system via the vagus nerve. The key regulatory receptor is the α7-subtype of the nicotinic acetylcholine receptor (α7nAChR), which is localized on the surface of the cells of immune system. CAP has been proved to be effective in suppressing the inflammation responses in acute lung injury (ALI). Dendritic cells (DCs), the important antigen-presenting cells (APCs), also express the α7nAChR. They not only play an important role in immune response priming but also in participating in the pathological process of ALI. Past studies have indicated that reducing the quantity of mature conventional DCs (cDCs) and inhibiting the maturation of pulmonary DCs may prove effective for the treatment of ALI. However, the effects of CAP on maturation, function and quantity of DCs and cDCs in ALI remain unclear. Objective It was hypothesized that the activation of CAP may inhibit the inflammatory response of ALI by regulating maturation, phenotype, and quantity of DCs and cDCs. This can be considered as an important intervention strategy for treating ALI. Methods GTS-21 (GTS-21 dihydrochloride), an α7nAchR agonist was administered in sepsis-induced ALI mice model and LPS-primed bone marrow-derived dendritic cells (BMDCs). The effects of GTS-21 were observed with respect to maturation, phenotype, and quantity of DCs, cDCs, and cDCs2 (type 2 cDCs), and the release of DC-related pro-inflammatory cytokines (such as IL-6, TNF-α, IL-18 IL-1β, IL-12p40, and HMGB1) in vivo and in vitro conditions. Results The results of the present study revealed that, GTS-21 treatment regulated the maturation of DCs and the production of DC-related pro-inflammatory cytokines in vitro and in sepsis-induced ALI mice model, it reduced the quantity of CD11c+MHCII+ cDCs and CD11c+CD11b+ cDCs2 in vivo experiment. Conclusions The activation of CAP contributes to the reduction in the inflammatory response in ALI by regulating maturation, phenotype, and quantity of DCs, cDCs, and cDCs2.

scholarly journals scRNA-Seq Identifies IL-1 Responsive Cell Subsets in the Skin Injury-induced Inflammatory Response

2020 ◽  
Vol 3 ◽  
Author(s):  
Kayla Harpold ◽  
Hong-Ming Zhou ◽  
Radomir Slominski ◽  
Leroy Seymour ◽  
Maria Bell ◽  
...  
Keyword(s):  
Wound Healing ◽  
Inflammatory Response ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Cell Populations ◽  
Skin Injury ◽  
Skin Wound Healing ◽  
Inflammatory Phase ◽  
Skin Biopsies

Inflammation is an integral aspect of skin wound healing; however, the mechanisms that regulate inflammatory cascades in this context are not well defined. To better understand how skin inflammation impacts wound healing, we developed an ex vivo skin culture system to model key aspects of the inflammatory phase of wound healing. In this model, a defined set of proinflammatory cytokines and chemokines, mirroring those produced in wounds in vivo, are produced when mouse or human skin biopsies are cultured ex vivo.  We refer to this pattern of cytokine and chemokine induction as the skin injury-induced inflammatory response. Previous studies in our laboratory demonstrated this response is initiated by the cytokine, interleukin 1 alpha (IL-1α). To understand the cellular sources and targets of IL-1α during the skin injury-induced inflammatory response, skin biopsies from mouse tail skin were cultured ex vivo for 8 hours followed by processing for single cell RNA sequencing (scRNAseq). Using bioinformatic software, R, and the package, Seurat, analysis of scRNAseq data from this experiment identified 22 distinct cell population clusters. While no populations exhibited significant expression of Il1a transcripts, multiple cell populations expressed Il1r1 transcripts, which encodes the ligand-specific subunit of the IL-1 receptor.  Notably, fibroblast, endothelial cell and stromal cell clusters were characterized by expression of Il1r1 and the skin injury-induced inflammatory response transcripts Il6, Cxcl1 and/or Csf3. Furthermore, Reactome Pathway Analysis suggested the Il-1 signaling axis was activated in these cell populations. This information provides a basis for future studies to understand how IL-1 signaling in fibroblasts, endothelial cells and stromal cells impacts wound healing in vivo, which could in turn lead to novel therapeutic approaches to clinically relevant outcomes.  

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