Fibroblast Cell Line Research Articles

Transcriptomic and proteomic strategies to reveal the mechanism of Gymnocypris przewalskii scale development

BackgroundFish scales are typical products of biomineralization and play an important role in the adaptation of fish to their environment. The Gymnocypris przewalskii scales are highly specialized, with scales embedded in only specific parts of the dermis, such as the areas around the anal fin and branchiostegite, making G. przewalskii an ideal material for biomineralization research. In this study, we aimed to unveil genes and pathways controlling scale formation through an integrated analysis of both transcriptome and proteome, of which G. przewalskii tissues of the dorsal skin (no scales) and the rump side skin (with scales) were sequenced. The sequencing results were further combined with cellular experiments to clarify the relationship between genes and signaling pathways.ResultsThe results indicated the following: (1) a total of 4,904 differentially expressed genes were screened out, including 3,294 upregulated genes and 1,610 downregulated genes (with a filtering threshold of |log2Fold-Change|> 1 and p-adjust < 0.05). The identified differentially expressed genes contained family members such as FGF, EDAR, Wnt10, and bmp. (2) A total of 535 differentially expressed proteins (DEPs) were filtered out from the proteome, with 204 DEPs downregulated and 331 DEPs upregulated (with a filtering threshold of |Fold-Change|> 1.5 and p < 0.05). (3) Integrated analyses of transcriptome and proteome revealed that emefp1, col1a1, col6a2, col16a1, krt8, and krt18 were important genes contributing to scale development and that PI3K-AKT was the most important signaling pathway involved. (4) With the use of the constructed G. przewalskii fibroblast cell line, emefp1, col1a1, col6a2, col16a1, krt8, and krt18 were confirmed to be positively regulated by the PI3K-AKT signaling pathway.ConclusionThis study provides experimental evidence for PI3K-AKT controlled scale development in G. przewalskii and would benefit further study on stress adaptation, scale biomineralization, and the development of skin appendages.

Influence of Ultrasound Stimulation on the Viability, Proliferation and Protein Expression of Osteoblasts and Periodontal Ligament Fibroblasts.

Among the adjunctive procedures to accelerate orthodontic tooth movement (OTM), ultrasound (US) is a nonsurgical form of mechanical stimulus that has been explored as an alternative to the currently available treatments. This study aimed to clarify the role of US in OTM by exploring different stimulation parameters and their effects on the biological responses of cells involved in OTM. Human fetal osteoblasts and periodontal ligament fibroblasts cell lines were stimulated with US at 1.0 and 1.5 MHz central frequencies and power densities of 30 and 60 mW/cm2 in continuous mode for 5 and 10 min. Cellular proliferation, metabolic activity and protein expression were analyzed. The US parameters that significantly improved the metabolic activity were 1.0 MHz at 30 mW/cm2 for 5 min and 1.0 MHz at 60 mW/cm2 for 5 and 10 min for osteoblasts; and 1.0 MHz at 30 mW/cm2 for 5 min and 1.5 MHz at 60 mW/cm2 for 5 and 10 min for fibroblasts. By stimulating with these parameters, the expression of alkaline phosphatase was maintained, while osteoprotegerin synthesis was induced after three days of US stimulation. The US stimulation improved the biological activity of both osteoblasts and periodontal ligament fibroblasts, inducing their osteogenic differentiation.

The Fatty Acid Composition and the MTT-Cytotoxicity Test of Commercially Available Commiphora Gileadensis Balsam Oil

Commiphora gileadensis, an aromatic plant, is traditionally used in Middle Eastern countries to prevent pain and inflammation. Fatty acid composition, peroxide number and free fatty acidity analyzes of commercially available C. gileadensis balsam oil and investigation of its effect on cell viability. Commercially available C. gileadensis balsam oil fatty acid composition, peroxide number and free fatty acidity values were determined by the IUPAC IID19 method, and MTT cell viability tests were performed on L929 fibroblast and HeLa (human epithelial cervical carcinoma) cell lines. Commercially available C. gileadensis balsam oil did not exert cytotoxic effects on both L929 fibroblast and HeLa (human epithelial cervical carcinoma) cell lines and promoted the growth of cell lines. Due to its growth promoting feature on L929 fibroblast cells, which is a healthy cell line, this material can be used as a cell culture medium additive.

Fusariotoxin-Induced Toxicity in Mesenchymal Stem Cells and Fibroblasts: A Comparison Between Differentiated and Undifferentiated Cells.

Humans are unknowingly exposed to mycotoxins through the consumption of plant-derived foods and processed products contaminated with these toxic compounds. In addition to agricultural losses, Fusarium toxins pose a threat to human health. However, the effects of fusariotoxins on the viability and proliferation of stem cells have not been fully explored. We investigated the cytotoxic effects of deoxynivalenol (DON) and B-trichothecene mix (MIX) on mesenchymal stem cells (MSCs) and the L929 fibroblast cell line. MSCs were isolated from the dental pulp tissue. The doubling time and viability of dental pulp stem cells (DPSCs) and L929 cells were determined using the MTT assay. The following doses of B-trichothecenes (0.25-16 µg/mL; 24 hours and 48 hours) were used to evaluate cytotoxicity. In addition, changes in the confluency-dependent response of DPSCs to DON toxicity were determined. Moreover, we investigated the effect of DON on cell death via acridine orange/ethidium bromide (AO/EB) double staining. A DON and MIX showed a dose- and time-dependent inhibitory effect on the proliferation of both cells. DPSCs exposed to DON for 48 hours (IC50 = 0.5 μg/mL) were found to be 16-fold more sensitive than L929 cells (IC50 = 8 μg/mL). Compared with a culture with 80% confluency, DPSCs from a 50% confluent culture were more sensitive to varying doses of DON (0.25-4 µg/mL, 24-48 hours). Moreover, AO/EB staining showed that treatment of DPSCs with DON led to a significant increase in cell death (17% for 2.4 µg/mL; 50% for 4.8 µg/mL). This study reveals that undifferentiated MSCs are significantly more sensitive to DON than differentiated somatic cells (L929). Given that humans are frequently exposed to these mycotoxins, our findings imply that prolonged exposure to them may also have harmful effects on cellular differentiation and embryonic development.

Cytotoxicity of Laser-Synthesized Nanoparticles of Elemental Bismuth.

High X-ray absorption combined with photothermal properties make bismuth nanoparticles (Bi NP) a promising agent for multimodal cancer theranostics. However, the synthesis of Bi NP by the "classical" chemical methods has numerous limitations, including potential toxicity of the produced nanomaterials. Here we studied in vitro toxicity of laser-synthesized Bi NP coated with Pluronic F-127 on mouse fibroblast cell line L929. The survival of L929 cells decreased linearly with increasing the concentration of Bi NP in a concentration range of 3-500 μg/ml; the LC50 value was 57 μg/ml. The unique combination of functional properties and moderate toxicity of the laser-synthesized Bi NP makes them a new promising platform for sensitization of multimodal cancer theranostics.

IN VITRO EVALUATION OF THE BIOLOGICAL ACTIVITY POTENTIAL OF ALOE VERA GEL: ANTIOXIDANT ACTIVITY AND CYTOTOXIC EFFECTS IN HEPG2 AND L929

Background: The place of natural and herbal products in alternative therapy is very important. A.vera is also an important plant that can be used in alternative therapy. Our study aimed to determine the antioxidant activity and cytotoxic effects of Aloe vera gel on HepG2 and L929 cell lines. Methods: The effects of A.vera on HepG2 and L929 were evaluated via the MTT analysis. The total antioxidant status and DPPH methods were used to determine the antioxidant capacity. Results: In our study, the mean cytotoxic effect on HepG2 of A.vera was determined to be 60.47 mg/mL at 24 hours and 44.64 mg/mL at 48 hours. In a healthy L929 fibroblast cell line, the best stimulating effect of A. vera was in 48 hours, and the EC50 value was calculated as 21.98±18.87 µg/ml. TAS was determined to be 0.2 mmol/L in A. vera. Free radical scavenging activity was determined at 21.58 mg/mL Conclusions: According to the obtained data, it was determined that A. vera gel has a concentration- and time-dependent cytotoxic effect on HepG2, while it has a proliferative effect on healthy cells. The fact that A. vera gel has antioxidant capacity, together with its positive effects on cells, shows that it can be an important support in treatments.

Salicylidene‐based homoleptic metal(II) complexes: Theoretical, in silico molecular modeling and in vitro cytotoxicity studies

A series of 12 salicylidene‐based homoleptic nickel(II), copper(II) and zinc(II) complexes with the general formula [M(L1–4)2] (1–12) containing the tridentate ligands 2‐(((2‐(([1,1′‐biphenyl]‐3‐ylmethylene)amino)phenyl)imino)methyl)phenol (HL1), 2‐(((2‐(([1,1′‐biphenyl]‐3‐ylmethylene)amino)phenyl)imino)methyl)‐4‐methoxyphenol (HL2), 2‐(((2‐(([1,1′‐biphenyl]‐3‐ylmethylene)amino)phenyl)imino)methyl)‐4‐ethoxyphenol (HL3) and 2‐(((2‐(([1,1′‐biphenyl]‐3‐ylmethylene)amino)phenyl)imino)methyl)‐4‐nitrophenol (HL4) have been synthesized and characterized by spectroscopic analysis. The tridentate nature of the ligands form an octahedral geometry around the central metal ion through the phenolic oxygen via deprotonation and two azomethine nitrogen atoms. Further, the charge transfer transitions taking place in the complexes have been explained using HOMO‐LUMO analysis. The in vitro cytotoxicity activity screened by WST assay against human metastatic (MDA‐MB‐231), hepatoma (HepG2) and colorectal adenocarcinoma (CaCo2) cancerous, and normal mouse fibroblast (NIH3T3) cell lines showed higher activity for the complexes 2, 6 and 10 with respect to the standard drug 5‐fluorouracil. The AO/EB, PI, Rh123 and DCFH‐DA staining results indicated that the complexes induced anticancer activity through apoptosis. Furthermore, the complexes interact with FGFR and P13Kγ kinase receptors through the hydrophobic, hydrogen bonding, π–π, σ–π, van der Waals and electrostatic interaction.

Immortalization of American miniature horse-derived fibroblast by cell cycle regulator with normal karyotype.

Immortalized cells serve as a crucial research tool that capitalizes on their robust proliferative properties for functional investigations of an organism. Establishing an immortalized American miniature horse cell line could yield valuable insights into these animals' genetic and physiological characteristics and susceptibility to health issues. To date, immortalized small horse cells with normal karyotypes have not been established. In this study, we successfully established primary and immortalized fibroblast cell lines through the combined expression of human-derived mutant cyclin-dependent kinase 4 (CDK4R24C), cyclin D1, and Telomerase Reverse Transcriptase (TERT), although CDK4R24C and cyclin D1, SV40T and TERT did not result in successful immortalization. Our comparison of the properties of these immortalized cells demonstrated that K4DT immortalized cells maintain a normal karyotype. Ultimately, our findings could pave the way for the development of targeted interventions to enhance the health and well-being of American miniature horses.

Pro-Fibrotic Effects of CCL18 on Human Lung Fibroblasts Are Mediated via CCR6.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.

Transcription Silencing and CpGs Hypermethylation as Therapeutic Gene Editing in Clinical Colorectal Adenocarcinoma Repression.

Colorectal cancer is the most common cancer in oncopathology, with an increasing incidence among the elderly during the last decade. Various genetic and environmental factors play important roles in the emergence of colorectal adenocarcinoma. Non-coding RNAs, approximately 20-22 nucleotides, are transcribed irregularly in many cancer cells and play a critical role in many metabolic pathways in clinical cancer cases. DNA methylation is a critical epigenetic alteration that controls gene expression. In the current study, transcriptional silencing and CpG hypermethylation were developed as a therapeutic gene editing strategy for the clinical repression of colorectal adenocarcinoma. A human colorectal adenocarcinoma cell line (Caco2) and a normal lung fibroblast cell line (Wi38) were utilized as the paradigms in this research to examine the effect of mir155 molecule transfection and CpGs-island (CGI) methylation. Cell counting was achieved using six-well and 24-well plates before transfection using a hemocytometer. The two cell lines were transfected with the mir155 agomir and antagomir molecules. The transfection efficiency, cell viability, cell IC50, and target gene expression were measured, and CGIs-methylation was achieved by bisulfate conversion. The outcomes revealed the downregulation of oncogenes (AKT1 and VCAM1 genes as cancer-associated genes) and the upregulation of tumor suppressor genes (TSGs, Tp53 and KEAP1). In addition, CpG-islands methylation showed significant blocking of the oncogene promoter regions, and the switch on of TSG promoter regions was continuous. miRNA-CGI-methylation led to the regression of Caco2 cell proliferation, suggesting the potential use of RNA silencing and DNA methylation in targeted gene therapy for colorectal cancer.

Skin Protection by Carotenoid Pigments.

Sunlight, despite its benefits, can pose a threat to the skin, which is a natural protective barrier. Phototoxicity caused by overexposure, especially to ultraviolet radiation (UVR), results in burns, accelerates photoaging, and causes skin cancer formation. Natural substances of plant origin, i.e., polyphenols, flavonoids, and photosynthetic pigments, can protect the skin against the effects of radiation, acting not only as photoprotectors like natural filters but as antioxidant and anti-inflammatory remedies, alleviating the effects of photodamage to the skin. Plant-based formulations are gaining popularity as an attractive alternative to synthetic filters. Over the past 20 years, a large number of studies have been published to assess the photoprotective effects of natural plant products, primarily through their antioxidant, antimutagenic, and anti-immunosuppressive activities. This review selects the most important data on skin photodamage and photoprotective efficacy of selected plant carotenoid representatives from in vivo studies on animal models and humans, as well as in vitro experiments performed on fibroblast and keratinocyte cell lines. Recent research on carotenoids associated with lipid nanoparticles, nanoemulsions, liposomes, and micelles is reviewed. The focus was on collecting those nanomaterials that serve to improve the bioavailability and stability of carotenoids as natural antioxidants with photoprotective activity.

P094 Lack of DNA methyltransferase DNMT3A in fibroblasts impairs cell growth and wound healing

Abstract Background Fibrotic scarring is an untreatable pathological wound-healing complication that invalidates correct organ function. However, pathomechanisms leading to intestinal fibrosis during inflammatory bowel disease (IBD) are not fully understood. Since the de novo DNA methyltransferase DNMT3A has been associated with increased risk for IBD and alters epithelial homeostasis, we sought to characterize how DNMT3A loss might affect fibroblasts, the key mesenchymal cells involved in wound healing. Methods We stably depleted Dnmt3a in 3T3 fibroblast cell line using CRISPR-Cas9 technology and subsequently selected GFP-positive clones using FACS sorting. Cell cultures were performed in vessels with or without 0.2% gelatin coat. Reverse transcription quantitative PCR, western blot, and immunofluorescence were used to confirm Dnmt3a gene deletion and characterize wild-type (WT) and knockout cells (KO). Wound healing assays in the presence or absence of tissue repair cytokine TGF-β served to quantify and assess cell capacity of wound closure. Results 3T3 fibroblasts lacking Dnmt3a acquire an activated myofibroblast phenotype (characterized by increased α-SMA expression) that also overexpress tissue remodelling matrix metalloproteinase Mmp3, and the inflammation-induced necroptosis marker RIPK3 when compared to their WT counterparts. In addition, Dnmt3a-depleted 3T3 cells grow overlapped in tightly aggregated foci (fig. 1), and when seeded in vessels without the gelatin coat, they form spheroids. To assess healing properties, the closure of a wound (gap) in a cell monolayer was monitored. While WT 3T3 fibroblasts closed the gap within 48h, Dnmt3a KO 3T3 cells were unable to refill the empty space and formed aggregated foci in the vicinity of the wound (fig. 1). When the wound healing assay was repeated in the presence of TGF-β, WT cells nearly closed the gap within 24h whereas Dnmt3a KO 3T3 still failed to grow. Conclusion We conclude that Dnmt3a is crucial for fibroblast homeostasis and correct wound healing. Furthermore, TGF-β signalling is impaired when Dnmt3a is lacking. Thus, additional characterization of Dnmt3a depletion in fibroblasts will deepen the understanding of fibrosis and point towards new therapeutic approaches for this untreatable complication.

The Characterization and Cytotoxic Evaluation of Chondrosia reniformis Collagen Isolated from Different Body Parts (Ectosome and Choanosome) Envisaging the Development of Biomaterials.

Chondrosia reniformis is a collagen-rich marine sponge that is considered a sustainable and viable option for producing an alternative to mammalian-origin collagens. However, there is a lack of knowledge regarding the properties of collagen isolated from different sponge parts, namely the outer region, or cortex, (ectosome) and the inner region (choanosome), and how it affects the development of biomaterials. In this study, a brief histological analysis focusing on C. reniformis collagen spatial distribution and a comprehensive comparative analysis between collagen isolated from ectosome and choanosome are presented. The isolated collagen characterization was based on isolation yield, Fourier-transformed infrared spectroscopy (FTIR), circular dichroism (CD), SDS-PAGE, dot blot, and amino acid composition, as well as their cytocompatibility envisaging the development of future biomedical applications. An isolation yield of approximately 20% was similar for both sponge parts, as well as the FTIR, CD, and SDS-PAGE profiles, which demonstrated that both isolated collagens presented a high purity degree and preserved their triple helix and fibrillar conformation. Ectosome collagen had a higher OHpro content and possessed collagen type I and IV, while the choanosome was predominately constituted by collagen type IV. In vitro cytotoxicity assays using the L929 fibroblast cell line displayed a significant cytotoxic effect of choanosome collagen at 2 mg/mL, while ectosome collagen enhanced cell metabolism and proliferation, thus indicating the latter as being more suitable for the development of biomaterials. This research represents a unique comparative study of C. reniformis body parts, serving as a support for further establishing this marine sponge as a promising alternative collagen source for the future development of biomedical applications.

In Vitro Release of Glycyrrhiza Glabra Extract by a Gel-Based Microneedle Patch for Psoriasis Treatment.

Microneedle patches are attractive drug delivery systems that give hope for treating skin disorders. In this study, to first fabricate a chitosan-based low-cost microneedle patch (MNP) using a CO2 laser cutter for in vitro purposes was tried and then the delivery and impact of Glycyrrhiza glabra extract (GgE) on the cell population by this microneedle was evaluated. Microscopic analysis, swelling, penetration, degradation, biocompatibility, and drug delivery were carried out to assess the patch's performance. DAPI staining and acridine orange (AO) staining were performed to evaluate cell numbers. Based on the results, the MNs were conical and sharp enough (diameter: 400-500 μm, height: 700-900 μm). They showed notable swelling (2 folds) during 5 min and good degradability during 30 min, which can be considered a burst release. The MNP showed no cytotoxicity against fibroblast cell line L929. It also demonstrated good potential for GgE delivery. The results from AO and DAPI staining approved the reduction in the cell population after GgE delivery. To sum up, the fabricated MNP can be a useful recommendation for lab-scale studies. In addition, a GgE-loaded MNP can be a good remedy for skin disorders in which cell proliferation needs to be controlled.

Towards label-free non-invasive autofluorescence multispectral imaging for melanoma diagnosis.

This study focuses on the use of cellular autofluorescence which visualizes the cell metabolism by monitoring endogenous fluorophores including NAD(P)H and flavins. It explores the potential of multispectral imaging of native fluorophores in melanoma diagnostics using excitation wavelengths ranging from 340 nm to 510 nm and emission wavelengths above 391 nm. Cultured immortalized cells are utilized to compare the autofluorescent signatures of two melanoma cell lines to one fibroblast cell line. Feature analysis identifies the most significant and least correlated features for differentiating the cells. The investigation successfully applies this analysis to pre-processed, noise-removed images and original background-corrupted data. Furthermore, the applicability of distinguishing melanomas and healthy fibroblasts based on their autofluorescent characteristics is validated using the same evaluation technique on patient cells. Additionally, the study tentatively maps the detected features to underlying biological processes. This research demonstrates the potential of cellular autofluorescence as a promising tool for melanoma diagnostics.

Aqueous distillate of mature leaves of Vernonia zeylanica (L.) Less. and Mallotus repandus (Rottler) Müll. Arg. cued from traditional medicine exhibits rapid wound healing properties

Ethnopharmacological relevanceSri Lankan traditional medicine uses Vernonia zeylanica and Mallotus repandus broadly for the treatment of a multitude of disease conditions, including wound healing. Aim of the studyWe aimed to scientifically validate the safety and efficacy of wound healing of an aqueous distillate of Vernonia zeylanica and Mallotus repandus (ADVM) mature leaves, tested on primary human dermal fibroblasts. Materials and methodsHuman dermal fibroblasts isolated from clinical waste from circumcision surgery were characterized by flowcytometry and trilineage differentiation. The MTT dye reduction assay, and the ex vivo wound healing scratch assay established wound healing properties of ADVM using the primary human dermal fibroblast cell line. Upregulation of genes associated with wound healing (MMP3, COL3A1, TGFB1, FGF2) were confirmed by RT qPCR. GC-MS chromatography evaluated the phytochemical composition of ADVM. ResultsCompared to the synthetic stimulant, β fibroblast growth factor, ADVM at 0.25% concentration on the primary dermal fibroblast cell line exhibited significant ex vivo, (i) 1.7-fold % cell viability (178.7% vs 304.3 %, p < 0.001), (ii) twofold greater % wound closure (%WC) potential (47.74% vs 80.11%, p < 0.001), and (iii) higher rate of % WC (3.251 vs 3.456 % WC/h, p < 0.05), sans cyto-genotoxicity. Up regulated expression of FGF2, TGFB1, COL3A1 and MMP3, genes associated with wound healing, confirmed effective stimulation of pathways of the three overlapping phases of wound healing (P < 0.05). GC-MS profile of ADVM characterized four methyl esters, which may be posited as wound healing phytochemicals. ConclusionsExceeding traditional medicine claims, the exvivo demonstration of rapid skin regeneration, reiterated by upregulated expression of genes related to wound healing pathways, sans cytotoxicity, propounds ADVM, cued from traditional medicine, as a potential safe and effective natural stimulant for rapid wound-healing. Additionally, it may serve as an effective proliferative stimulant of dermal fibroblasts for cell therapy, with potential in reparative and regenerative therapy of skin disorders.

Coprecipitated Keratin/hydroxyapatite nanocomposites assisted by microwave/ultrasound irradiation, and its cytotoxic evaluation on NIH/3T3 fibroblast cells

This work is aimed to synthesize Hydroxyapatite (Hap) and Keratin/Hydroxyapatite (KerHap) nanocomposites by coprecipitation using the simultaneous microwave/ultrasound (Mw/Us) method to confer specific textural and morphological properties to the materials using keratin (Ker) extracted from human hair. Four materials were synthesized applying different powers of Mw/Us irradiation: Hap (1100 W Mw, 40 kHz Us), KerHap A (700 W Mw, 40 kHz Us), KerHap B (900 W Mw, 40 kHz Us) and KerHap C (1100 W Mw, 40 kHz Us) at 120 °C for 10 min. The materials were characterized according to their structure (FTIR, XRD, Raman spectroscopy), morphology and size (TEM), and thermal properties (TG). Their cytotoxic effects were studied at different concentrations (25, 50, 100, 200, and 400 μg/mL) using the fibroblast cell line (NIH/3T3) by MTT assay at 24, 48, and 72 h. The Ker extract exhibited a major molecular weight band between 11–10 kDa in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Morphology, particle, and crystallite sizes were dependent on the Mw irradiation power applied and keratin added during the synthesis. The MTT assay at 48 h demonstrated a significant increase in cell viability of 39% for Hap at 50 µg/mL, for KerHap A did not show a significant difference, while KerHap B-C showed a significant increase at 200 µg/mL of 30% in cell viability. It means that fibroblast presented better cell adhesion on Hap than on KerHap A-C composites; it may be due to keratin blocking Hap pores because of electrostatic interactions between keratin and Hap.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species

BackgroundThe gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes.MethodsIn this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed.ResultsThis novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines.ConclusionsAn AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite–host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.Graphical

Abstract A021: Adjuvant iC9.B7-H3 CAR T cell-based immunotherapy effectively eradicates local and distant metastases in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Despite negative margin resections of the cancer and systemic therapy, PDAC often recurs. Pancreatic cancer-associated fibroblasts (CAFs) contribute to a highly desmoplastic tumor microenvironment (TME), preventing T cell infiltration. Moreover, CAF-secreted factors suppress the anti-tumor activity of T cells and shift tumor cells toward invasive proliferative and mesenchymal phenotypes. Therefore, targeting CAFs can enhance T-cell infiltration and improve treatment outcomes. In this study, we investigate using Chimeric Antigen Receptor (CAR) T cells targeting B7-H3 to eliminate PDAC metastatic disease in a preclinical mouse model. B7-H3, an immune checkpoint molecule, is highly expressed on PDAC cells and CAFs. Our CART-B7-H3 construct includes an inducible caspase 9 (iC9) suicide gene, which allows for the controlled elimination of the CAR T cells in the event of overwhelming cytokine release syndrome. Methods: The efficacy of iC9.B7-H3 CAR T cells was tested in vitro against human PDAC and fibroblast cell lines at various ratios. The antitumor activity of iC9.B7-H3 CAR T cells was assessed in PDAC cells after exposure to CAF-conditioned media, simulating TME with immunosuppressive cytokines. To validate the therapeutic potential of iC9.B7-H3 CAR T cells in vivo, NSG mice were orthotopically engrafted with patient-derived PDAC6 cells and hCAF1 cells at a ratio of 1:9, reflecting a clinically relevant model. After three weeks, the mice underwent surgical resection of the primary tumor (distal pancreatectomy and splenectomy) and were subsequently treated systemically with iC9.B7-H3 CAR T cells. Results: In vitro, at a high effector-to-target (E:T) ratio, iC9.B7-H3 CAR T cells effectively eliminated PDAC cells and CAFs. However, their anti-tumor activity was diminished when the E:T ratio was lowered (PDAC6 tumor cell killing (TCK): 92±5% at 1:1, hCAF1 TCK: 81.67±5% at 1:1, compared to PDAC6 TCK: 26±5% at 1:10, hCAF1 TCK: 0% at 1:10, P&amp;lt;0.0001). PDAC cells co-cultured with CAF cells demonstrated reduced susceptibility to elimination by iC9.B7-H3 CAR T cells compared to PDAC cells alone (PDAC3 TCK: 92±5% at 1:1, PDAC3+hCAF1 (1:9 ratio) TCK: 81±5% at 1:1, P=0.002). However, incubation with CAF-conditioned media did not significantly affect the elimination of PDAC cells by iC9.B7-H3 CAR T cells, which may suggest that CAFs may require direct cell-cell contact to exert their effects. In vivo, iC9.B7-H3 CAR T cells effectively eradicated both local and distant PDAC metastases for the length of the experiment (160 days), indicating long-term efficacy in the treatment of PDAC metastases with no adverse side effects. Conclusion: These data suggest that iC9.B7-H3 CAR T cells may be an effective adjuvant treatment for the eradication of micro-metastases and the prevention of disease recurrence in PDAC patients. Citation Format: Shahrzad Arya, Seyed Amir Sanatkar, Cristina Martin, Marco Ventin, Jingyu Jia, Giulia Cattaneo, Gabriella Lionetto, David T. Ting, Xinhui Wang, Sandra Ryeom, Cristina R. Ferrone, Soldano Ferrone. Adjuvant iC9.B7-H3 CAR T cell-based immunotherapy effectively eradicates local and distant metastases in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A021.

ROS-responsive electrospun poly(amide thioketal) mats for wound dressing applications

Excessive levels of reactive oxygen species (ROS) are usually associated with the inflammatory response during the wound healing process. Therefore, strategies that can locally control the adverse effects of excessive formation of ROS may lead to improved healing times. In this work, ROS-responsive scavenging materials were developed using a degradable poly(amide thioketal) (PATK) polymer. This PATK polymer was prepared using a thioketal (TK) diacid monomer which gives the polymer the ability to be degraded by ROS. This PATK polymer was used to prepare electrospun mats by blending it with the biocompatible poly(ε-caprolactone) (PCL). The developed mats showed good degradability when exposed to an oxidative environment and can also be degraded by a photo-mediated mechanism. Cytotoxicity tests performed in vitro on a fibroblast cell line showed that the mats had no adverse effects on the cells, confirming the potential of these systems for biomedical applications. In addition, the antioxidant capacity of the membranes was also found to be good, which could be relevant for wound dressing applications.