Evaluation of bacteriophages for the alleviation of potential bacterial contamination risks in developmental engineering.

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE.

Spatial transcriptomic sequencing reveals immune microenvironment features of Mycobacterium tuberculosis granulomas in lung and omentum.

Granulomas are a key pathological feature of tuberculosis (TB), characterized by cell heterogeneity, spatial composition, and cellular interactions, which play crucial roles in granuloma progression and host prognosis. This study aims to analyze the transcriptome profiles of cell populations based on their spatial location and to understand the core transcriptome characteristics of granuloma formation and development. Methods In this study, we collected four clinical biopsy samples including Mycobacterium tuberculosis (Mtb) infected lung (MTB-L) and omentum tissues (MTB-O), as well as two lung and omentum biopsies from non-TB patients. The tissues were analyzed by spatial transcriptomics to create a spatial atlas. Utilizing cell enrichment scores and intercellular communication analysis, we investigated the transcriptome signatures of cell populations in various spatial regions and identified genes that may play a decisive role in the formation of pulmonary and omental tuberculosis granulomas. To validate our major findings, an in vitro TB model based on organoid-macrophage co-culture was established. Results Spatial transcriptomics mapped the cell composition and spatial distribution characteristics of tuberculosis granulomas in lung and omental tissues infected with Mtb. The characteristics and evolutionary relationships of major cell populations in granulomas reveal a shift in the immune microenvironment: from a predominance of B cells and fibroblasts in pulmonary granulomas to a predominance of myeloid cells and fibroblasts in omental granulomas. Furthermore, our data identified key differentially expressed genes across cell clusters and regions, showing that upregulation of collagen genes is a common feature of granulomas. Using an organoid-macrophage co-culture model, we demonstrated the notable efficacy of Thrombospondin-1 (THBS1) in reducing protein expression levels related to extracellular matrix remodeling. Conclusion These results provide insights into the pathogenesis and development of tuberculosis, enhancing our understanding of the composition and interactions of tuberculosis granuloma cells from a spatial perspective, and pave the way for novel adjuvant treatments for tuberculosis.

Development of a Coculture Model for Assessing Competing Host Mammalian Cell and Bacterial Attachment on Zirconia versus Titanium.

Objectives: Coculture models are limited by bacteria rapidly outcompeting host mammalian cells for nutrients in vitro, resulting in mammalian cell death. The goal of this study was to develop a coculture model enabling survival of mammalian cells and oral bacterial species to assess their competition for growth on dental implant materials. Methods: Two early colonizing oral bacterial species, Streptococcus mutans or Actinomyces naeslundii, were grown in coculture with primary human macrophages or human gingival fibroblasts for up to 7 days on tissue-culture treated polystyrene or polished titanium and zirconia disks. Chloramphenicol was supplemented in cell culture medium at bacteriostatic concentrations to maintain stable bacterial inoculum size. Planktonic and adherent bacterial growth was assessed via spot plating while mammalian cell growth and attachment were evaluated using colorimetric metabolic assay and confocal fluorescence microscopy, respectively. Results: Macrophages and fibroblasts proliferated in the presence of S. mutans and maintained viability above 70% during coculture for up to 7 days on tissue-culture treated polystyrene and polished titanium and zirconia. In contrast, both mammalian cell types exhibited decreasing proliferation and surface coverage on titanium and zirconia over time in coculture with A. naeslundii versus control. S. mutans and A. naeslundii were maintained within an order of magnitude of seeding inoculum sizes throughout coculture. Significance: Cell culture medium supplemented with antibiotics at bacteriostatic concentrations can suppress bacterial overgrowth and facilitate mammalian cell viability in coculture model systems. Within the study's limitations, oral bacteria and mammalian cell growth in coculture are comparable on polished titanium and zirconia surfaces.

Stable in vitro fluorescence for enhanced live imaging of infection models for Batrachochytrium dendrobatidis.

Realistic and modifiable infection models are required to study the pathogenesis of amphibian chytridiomycosis. Understanding the mechanism by which Batrachochytrium dendrobatidis (Bd) can infect and kill diverse amphibians is key to mitigating this pathogen and preventing further loss of biodiversity. In vitro studies of Bd typically rely on a tryptone based growth media, whereas the recent development of a kidney cell-line infection model has provided a more realistic alternative, without the need for live animals. Here we use expression of a fluorescent reporter to enhance the in vitro cell-line based growth assay, and show that transformed Bd cells are able to invade and grow in an amphibian kidney epithelial cell line (A6) as well as in a new system using a lung fibroblast cell line (DWJ). Both Bd and host cells were modified to express reporter fluorescent proteins, enabling immediate and continuous observation of the infection process without the need for destructive sampling for fixation and staining. Plasmid DNA conferring hygromycin resistance and TdTomato (RFP) expression was delivered to Bd zoospores via electroporation, and continuous antibiotic selection after recovery produced stable fluorescent Bd transformants. Host cells (A6 and DWJ) were transfected before each assay using lipofection to deliver plasmid DNA conferring green fluorescent protein (GFP) and containing an empty shRNA expression cassette. Bd RFP expression allowed easy localisation of fungal cells and identification of endobiotic growth was assisted by host GFP expression, by allowing visualization of the space in the host cell occupied by the invading fungal body. In addition to enabling enhanced live imaging, these methods will facilitate future genetic modification and characterisation of specific genes and their effect on Bd virulence.

Control of pathogenic bacterial biofilm associated with acne and the anti-inflammatory potential of an essential oil blend.

Acne is one of the most common skin conditions worldwide, with multifactorial origins it affects areas of the skin with hair follicles and sebaceous glands that become clogged. Bacterial incidence aggravates treatment due to resistance to antimicrobial agents and production of virulence factors such as biofilm formation. Based on these information, this study aims to conduct in vitro evaluations of the antibacterial activity of essential oils (EOs), alone and in combination, against Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis in planktonic and biofilm forms. This study also assessed the anti-inflammatory potential (TNF-α) and the effects of EOs on the viability of human keratinocytes (HaCaT), murine fibroblasts (3T3-L1), and bone marrow-derived macrophages (BMDMs). Of all EOs tested, 13 had active action against P. acnes, 9 against S. aureus, and 9 against S. epidermidis at concentrations of 0.125-2.0 mg/mL. Among the most active plant species, a blend of essential oil (BEOs) was selected, with Cymbopogon martini (Roxb.) Will. Watson, Eugenia uniflora L., and Varronia curassavica Jacq., the latter due to its anti-inflammatory action. This BEOs showed higher inhibition rates when compared to chloramphenicol against S. aureus and S. epidermidis, and higher eradication rates when compared to chloramphenicol for the three target species. The BEOs did not affect the cell viability of cell lines evaluated, and the levels of TNF-α decreased. According to these results, the BEOs evaluated showed potential for the development of an alternative natural formulation for the treatment of acne.

Staphylococcus aureus adapts to exploit collagen-derived proline during chronic infection.

Staphylococcus aureus is a pulmonary pathogen associated with substantial human morbidity and mortality. As vaccines targeting virulence determinants have failed to be protective in humans, other factors are likely involved in pathogenesis. Here we analysed transcriptomic responses of human clinical isolates of S. aureus from initial and chronic infections. We observed upregulated collagenase and proline transporter gene expression in chronic infection isolates. Metabolomics of bronchiolar lavage fluid and fibroblast infection, growth assays and analysis of bacterial mutant strains showed that airway fibroblasts produce collagen during S. aureus infection. Host-adapted bacteria upregulate collagenase, which degrades collagen and releases proline. S. aureus then imports proline, which fuels oxidative metabolism via the tricarboxylic acid cycle. Proline metabolism provides host-adapted S. aureus with a metabolic benefit enabling out-competition of non-adapted strains. These data suggest that clinical settings characterized by airway repair processes and fibrosis provide a milieu that promotes S. aureus adaptation and supports infection.

Decoding host cell interaction- and fluconazole-induced metabolic alterations and drug resistance in Candida auris.

Candida auris is an emerging drug-resistant pathogen associated with high mortality rates. This study aimed to explore the metabolic alterations and associated pathogenesis and drug resistance in fluconazole-treated Candida auris-host cell interaction. Compared with controls, secreted metabolites from fluconazole-treated C. auris and fluconazole-treated C. auris-host cell co-culture demonstrated notable anti-Candida activity. Fluconazole caused significant reductions in C. auris cell numbers and aggregated phenotype. Metabolites produced by C. auris with potential fungal colonization, invasion, and host immune evasion effects were identified. Metabolites known to enhance biofilm formation produced during C. auris-host cell interaction were inhibited by fluconazole. Fluconazole enhanced the production of metabolites with biofilm inhibition activity, including behenyl alcohol and decanoic acid. Metabolites with potential Candida growth inhibition activity such as 2-palmitoyl glycerol, 1-tetradecanol, and 1-nonadecene were activated by fluconazole. Different patterns of proinflammatory cytokine expression presented due to fluconazole concentration and host cell type (fibroblasts versus macrophages). This highlights the immune response's complexity, emphasizing the necessity for additional research to comprehend cell-type-specific responses to antifungal therapies. Both host cell interaction and fluconazole treatment increased the expression of CDR1 and ERG11 genes, both associated with drug resistance. This study provides insights into pathogenesis in C. auris due to host cell interaction and fluconazole treatment. Understanding these interactions is crucial for enhancing fluconazole sensitivity and effectively combating C. auris.

Decoding the complexity of delayed wound healing following Enterococcus faecalis infection.

Wound infections are highly prevalent and can lead to delayed or failed healing, causing significant morbidity and adverse economic impacts. These infections occur in various contexts, including diabetic foot ulcers, burns, and surgical sites. Enterococcus faecalis is often found in persistent non-healing wounds, but its contribution to chronic wounds remains understudied. To address this, we employed single-cell RNA sequencing (scRNA-seq) on infected wounds in comparison to uninfected wounds in a mouse model. Examining over 23,000 cells, we created a comprehensive single-cell atlas that captures the cellular and transcriptomic landscape of these wounds. Our analysis revealed unique transcriptional and metabolic alterations in infected wounds, elucidating the distinct molecular changes associated with bacterial infection compared to the normal wound healing process. We identified dysregulated keratinocyte and fibroblast transcriptomes in response to infection, jointly contributing to an anti-inflammatory environment. Notably, E. faecalis infection prompted a premature, incomplete epithelial-mesenchymal transition in keratinocytes. Additionally, E. faecalis infection modulated M2-like macrophage polarization by inhibiting pro-inflammatory resolution in vitro, in vivo, and in our scRNA-seq atlas. Furthermore, we discovered macrophage crosstalk with neutrophils, which regulates chemokine signaling pathways, while promoting anti-inflammatory interactions with endothelial cells. Overall, our findings offer new insights into the immunosuppressive role of E. faecalis in wound infections.

If wounds get infected, they heal much more slowly, sometimes leading to skin damage and other complications, including disseminated infections or even amputation. Infections can happen in many types of wounds, ranging from ulcers in patients with diabetes to severe burns. If infections are not cleared quickly, the wounds can become 'chronic' and are unable to heal without intervention. Enterococcus faecalis is a type of bacteria that normally lives in the gut. Within that environment, in healthy people, it is not harmful. However, if it comes into contact with wounds ­ particularly diabetic ulcers or the site of a surgery ­ it can cause persistent infections and prevent healing. Although researchers are beginning to understand how E. faecalis initially colonises wounds, the biological mechanisms that transform these infections into chronic wounds are still largely unknown. Celik et al. therefore set out to investigate exactly how E. faecalis interferes with wound healing. To do this, Celik et al. looked at E. faecalis-infected wounds in mice and compared them to uninfected ones. Using a genetic technique called single-cell RNA sequencing, Celik et al. were able to determine which genes were switched on in individual skin and immune cells at the site of the wounds. This in turn allowed the researchers to determine how those cells were behaving in both infected and uninfected conditions. The experiments revealed that when E. faecalis was present in wounds, several important cell types in the wounds did not behave normally. For example, although the infected skin cells still underwent a change in behaviour required for healing (called an epithelial-mesenchymal transition), the change was both premature and incomplete. In other words, the skin cells in infected wounds started changing too early and did not finish the healing process properly. E. faecalis also changed the way macrophages and neutrophils worked within the wounds. These are cells in our immune system that normally promote inflammation, a process involved in both uninfected wounds or during infections and is a key part of wound healing when properly controlled. In the E. faecalis-infected wounds, these cells' inflammatory properties were suppressed, making them less helpful for healing. These results shed new light on how E. faecalis interacts with skin cells and the immune system to disrupt wound healing. Celik et al. hope that this knowledge will allow us to find new ways to target E. faecalis infections, and ultimately develop treatments to help chronic wounds heal better and faster.

Antimicrobial efficacy of direct air gas soft jet plasma for the in vitro reduction of oral bacterial biofilms.

The aim of this study was to evaluate the antimicrobial efficacy of an air gas soft jet CAP for its potential use in removing oral biofilms, given that plasma-based technologies have emerged as promising methods in periodontology. Two types of biofilms were developed, one by Streptococcus mutans UA 159 bacterial strain and the other by a complex mixture of saliva microorganisms isolated from a patient with periodontitis. This latter biofilm was characterized via Next Generation Sequencing to determine the main bacterial phyla. The CAP source was applied at a distance of 6 mm for different time points. A statistically significant reduction of both CFU count and XTT was already detected after 60 s of CAP treatment. CLSM analysis supported CAP effectiveness in killing the microorganisms inside the biofilm and in reducing the thickness of the biofilm matrix. Cytotoxicity tests demonstrated the possible use of CAP without important side effects towards human gingival fibroblasts cell line. The current study showed that CAP treatment was able to significantly reduce preformed biofilms developed by both S. mutans and microorganisms isolated by a saliva sample. Further studies should be conducted on biofilms developed by additional saliva donors to support the potential of this innovative strategy to counteract oral pathogens responsible for periodontal diseases.

Chondrocyte Invasion May Be a Mechanism for Persistent Staphylococcus Aureus Infection In Vitro.

BACKGROUND: Recurrent bone and joint infection with Staphylococcus aureus is common. S. aureus can invade and persist in osteoblasts and fibroblasts, but little is known about this mechanism in chondrocytes. If S. aureus were able to invade and persist within chondrocytes, this could be a difficult compartment to treat. QUESTION/PURPOSE: Can S. aureus infiltrate and persist intracellularly within chondrocytes in vitro? METHODS: Cell lines were cultured in vitro and infected with S. aureus. Human chondrocytes (C20A4) were compared with positive controls of human osteoblasts (MG63) and mouse fibroblasts (NIH3T3), which have previously demonstrated S. aureus invasion and persistence (human fibroblasts were not available to us). Six replicates per cell type were followed for 6 days after infection. Cells were treated daily with antibiotic media for extracellular killing. To determine whether S. aureus can infiltrate chondrocytes, fluorescence microscopy was performed to qualitatively assess the presence of intracellular bacteria, and intracellular colony-forming units (CFU) were enumerated 2 hours after infection. To determine whether S. aureus can persist within chondrocytes, intracellular CFUs were enumerated from infected host cells each day postinfection. RESULTS: S. aureus invaded human chondrocytes (C20A4) at a level (2.8 x 10 5 ± 5.5 x 10 4 CFUs/mL) greater than positive controls of human osteoblasts (MG63) (9.5 x 10 2 ± 2.5 x 10 2 CFUs/mL; p = 0.01) and mouse fibroblasts (NIH3T3) (9.1 x 10 4 ± 2.5 x 10 4 CFUs/mL; p = 0.02). S. aureus also persisted within human chondrocytes (C20A4) for 6 days at a level (1.4 x 10 3 ± 5.3 x 10 2 CFUs/mL) greater than that of human osteoblasts (MG63) (4.3 x 10 2 ± 3.5 x 10 1 CFUs/mL; p = 0.02) and mouse fibroblasts (NIH3T3) (0 CFUs/mL; p < 0.01). S. aureus was undetectable within mouse fibroblasts (NIH3T3) after 4 days. There were 0 CFUs yielded from cell media, confirming extracellular antibiotic treatment was effective. CONCLUSION: S. aureus readily invaded human chondrocytes (C20A4) in vitro and persisted viably for 6 days after infection, evading extracellular antibiotics. Chondrocytes demonstrated a greater level of intracellular invasion and persistence by S. aureus than positive control human osteoblast (MG63) and mouse fibroblast (NIH3T3) cell lines. CLINICAL RELEVANCE: Chondrocyte invasion and persistence may contribute to recurrent bone and joint infections. Additional research should assess longer periods of persistence and whether this mechanism is present in vivo.

Isolation, characterization, and fibroblast uptake of bacterial extracellular vesicles from Porphyromonas gingivalis strains.

Periodontitis is an inflammatory condition caused by bacteria and represents a serious health problem worldwide as the inflammation damages the supporting tissues of the teeth and may predispose to systemic diseases. Porphyromonas gingivalis is considered a keystone periodontal pathogen that releases bacterial extracellular vesicles (bEVs) containing virulence factors, such as gingipains, that may contribute to the pathogenesis of periodontitis. This study aimed to isolate and characterize bEVs from three strains of P. gingivalis, investigate putative bEV uptake into human oral fibroblasts, and determine the gingipain activity of the bEVs. bEVs from three bacterial strains, ATCC 33277, A7A1-28, and W83, were isolated through ultrafiltration and size-exclusion chromatography. Vesicle size distribution was measured by nano-tracking analysis (NTA). Transmission electron microscopy was used for bEV visualization. Flow cytometry was used to detect bEVs and gingipain activity was measured with an enzyme assay using a substrate specific for arg-gingipain. The uptake of bEVs into oral fibroblasts was visualized using confocal microscopy. NTA showed bEV concentrations from 108 to 1011 particles/mL and bEV diameters from 42 to 356 nm. TEM pictures demonstrated vesicle-like structures. bEV-gingipains were detected both by flow cytometry and enzyme assay. Fibroblasts incubated with bEVs labeled with fluorescent dye displayed intracellular localization consistent with bEV internalization. In conclusion, bEVs from P. gingivalis were successfully isolated and characterized, and their uptake into human oral fibroblasts was documented. The bEVs displayed active gingipains demonstrating their origin from P. gingivalis and the potential role of bEVs in periodontitis.

Cell culture media dependent in vitro dynamics and culture characteristics of adult caprine dermal fibroblast cells.

The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.

The Role of Gingival Fibroblasts in the Pathogenesis of Periodontitis.

Gingival fibroblasts (GFs) are essential components of the periodontium, which are responsible for the maintenance of tissue structure and integrity. However, the physiological role of GFs is not restricted to the production and remodeling of the extracellular matrix. GFs also act as sentinel cells that modulate the immune response to oral pathogens invading the gingival tissue. As an important "nonclassical" component of the innate immune system, GFs respond to bacteria and damage-related signals by producing cytokines, chemokines, and other inflammatory mediators. Although the activation of GFs supports the elimination of invading bacteria and the resolution of inflammation, their uncontrolled or excessive activation may promote inflammation and bone destruction. This occurs in periodontitis, a chronic inflammatory disease of the periodontium initiated and sustained by dysbiosis. In the inflamed gingival tissue, GFs acquire imprinted proinflammatory phenotypes that promote the growth of inflammophilic pathogens, stimulate osteoclastogenesis, and contribute to the chronicity of inflammation. In this review, we discuss the biological functions of GFs in healthy and inflamed gingival tissue, highlighting recent studies that provide insight into their role in the pathogenesis of periodontal diseases. We also draw parallels with the recently discovered fibroblast populations identified in other tissues and their roles in health and disease. This knowledge should be used in future studies to discover more about the role of GFs in periodontal diseases, especially chronic periodontitis, and to identify therapeutic strategies targeting their pathological interactions with oral pathogens and the immune system.

Gut microbiota modulates lung fibrosis severity following acute lung injury in mice.

Independent studies demonstrate the significance of gut microbiota on the pathogenesis of chronic lung diseases; yet little is known regarding the role of the gut microbiota in lung fibrosis progression. Here we show, using the bleomycin murine model to quantify lung fibrosis in C57BL/6 J mice housed in germ-free, animal biosafety level 1 (ABSL-1), or animal biosafety level 2 (ABSL-2) environments, that germ-free mice are protected from lung fibrosis, while ABSL-1 and ABSL-2 mice develop mild and severe lung fibrosis, respectively. Metagenomic analysis reveals no notable distinctions between ABSL-1 and ABSL-2 lung microbiota, whereas greater microbial diversity, with increased Bifidobacterium and Lactobacilli, is present in ABSL-1 compared to ABSL-2 gut microbiota. Flow cytometric analysis reveals enhanced IL-6/STAT3/IL-17A signaling in pulmonary CD4 + T cells of ABSL-2 mice. Fecal transplantation of ABSL-2 stool into germ-free mice recapitulated more severe fibrosis than transplantation of ABSL-1 stool. Lactobacilli supernatant reduces collagen 1 A production in IL-17A- and TGFß1-stimulated human lung fibroblasts. These findings support a functional role of the gut microbiota in augmenting lung fibrosis severity.

Inflammasome dysregulation in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: Uncontrolled production of Interleukin-1ß (IL-1ß), a major proinflammatory cytokine, is associated with tissue destruction in periodontal disease. IL-1ß production is controlled by inflammasomes which are multiprotein regulatory complexes. The current study aimed to elucidate potential regulatory pathways by monitoring the effects of periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) on inflammasomes and their regulators in human gingival fibroblasts (HGFs) in vitro. METHODS: HGFs were exposed to Fn and Pg alone or in combination for 24 hr at a multiplicity of infection of 100, ±30 min exposure with 5 mM adenosine triphosphate (ATP) incubation. Gene expression of NLRP3 and AIM2, inflammasome regulatory proteins POP1, CARD16 and TRIM16, and inflammasome components ASC and CASPASE 1, and IL-1ß, were evaluated by RT-PCR. Pro- and mature IL-1ß levels were monitored intracellularly by immunocytochemistry and extracellularly by ELISA. RESULTS: Fn + ATP significantly upregulated NLRP3, AIM2, IL-1ß, ASC, and CASPASE 1; however, it downregulated POP1 and TRIM16. Pg + ATP downregulated NLRP3, ASC, POP1, but upregulated IL-1ß and CARD16. Pg + Fn+ATP significantly upregulated AIM2, IL-1ß and CARD16, and downregulated POP1, TRIM16, and CASPASE 1. Pg + ATP exposure significantly increased pro- and mature IL-1ß production. CONCLUSION: Bacterial exposure with ATP may deregulate IL-1ß by dysregulating inflammasomes and their regulators in HGFs.

Cytotoxicity Comparison of a Calcium Silicate-Based Resin Cement versus Conventional Self-Adhesive Resin Cement and a Resin-Modified Glass Ionomer: Cell Viability Analysis

Abstract Objective: To compare the cytotoxicity level of a new calcium silicate-based resin cement (TheraCem) with two commonly used cements, including a conventional self-adhesive resin cement (Panavia SA) and a resinmodified glass ionomer cement (FujiCem2), on the human gingival fibroblast cells after 24 and 48 hours. Material and Methods: Twelve discs of each cement type were fabricated. The extract of cement disks was made by incubating them in the cell medium. Human gingival fibroblast cells were cultured and exposed to cement extracts for 24 h and 48 h. MTT assay was performed on extracts and optical density and cell viability rates were calculated by the spectrophotometer device at 570 nm. Data were analyzed using ANOVA and Tukey HSD tests. Results: The cell viability rates after 24 hours and 48 hours were as follows: TheraCem: 89.24% and 85.46%, Panavia SA: 49.51% and 46.57% and FujiCem2: 50.63% and 47.36%. TheraCem represented the highest cell viability rate. However, no significant difference was noted between Panavia SA and FujiCem2. Time had no significant effect on cell viability. Conclusion: TheraCem exhibited the best results among three tested cements and was considered non-toxic. Panavia SA and FujiCem2 were not significantly different regarding the cell viability rate. Time had no significant effect on the cytotoxicity level of cements (AU).

Antimicrobial and cytotoxic activity of electrosprayed chitosan nanoparticles against endodontic pathogens and Balb/c 3T3 fibroblast cells.

The aims of this study were to synthesize highly positively charged chitosan nanoparticles (Ch-Np) using the electrospraying technique, and to test their antimicrobial activity against endodontic pathogens, and cytotoxicity against fibroblast cells. Ch-Np were synthesized from low molecular weight chitosan (LMW-Ch) using the electrospraying technique, and characterized. The antimicrobial activity was evaluated against Streptococcus mutans, Enterococcus faecalis, and Candida albicans in their planktonic state using a Time-Kill Test performed by using broth micro-dilution technique, and against biofilm biomass using a microtiter plate biofilm assay. The cytotoxicity was evaluated using Balb/c 3T3 fibroblast cells with the standard MTT assay. Electrospraying of LMW-Ch produced Ch-Np with an average size of 200 nm, and a surface charge of 51.7 mV. Ch-Np completely eradicated S. mutans and E. faecalis in the planktonic state and showed fungistatic activity against C. albicans. Furthermore, it significantly reduced the biofilm biomass for all the tested microbial species [S. mutans (p = 0.006), E. faecalis (p < 0.0001), and C. albicans (p = 0.004)]. When tested for cytotoxicity using 3T3 cells, Ch-Np showed no cytotoxicity. In conclusion, the highly positively charged, colloidal dispersion of Ch-Np are effective as a biocompatible endodontic antimicrobial agent.

Human Renal Fibroblasts, but Not Renal Epithelial Cells, Induce IL-1ß Release during a Uropathogenic Escherichia coli Infection In Vitro.

Understanding how uropathogenic Escherichia coli (UPEC) modulates the immune response in the kidney is essential to prevent UPEC from reaching the bloodstream and causing urosepsis. The purpose of this study was to elucidate if renal fibroblasts can release IL-1ß during a UPEC infection and to investigate the mechanism behind the IL-1ß release. We found that the UPEC strain CFT073 induced an increased IL-1ß and LDH release from renal fibroblasts, but not from renal epithelial cells. The UPEC-induced IL-1ß release was found to be NLRP3, caspase-1, caspase-4, ERK 1/2, cathepsin B and serine protease dependent in renal fibroblasts. We also found that the UPEC virulence factor α-hemolysin was necessary for IL-1ß release. Conditioned medium from caspase-1, caspase-4 and NLRP3-deficient renal fibroblasts mediated an increased reactive oxygen species production from neutrophils, but reduced UPEC phagocytosis. Taken together, our study demonstrates that renal fibroblasts, but not renal epithelial cells, release IL-1ß during a UPEC infection. This suggest that renal fibroblasts are vital immunoreactive cells and not only structural cells that produce and regulate the extracellular matrix.

HATMSC Secreted Factors in the Hydrogel as a Potential Treatment for Chronic Wounds-In Vitro Study.

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.

Mitochondrial DNA Haplogroup Related to the Prevalence of Helicobacter pylori.

Mitochondria are essential organelles that are not only responsible for energy production but are also involved in cell metabolism, calcium homeostasis, and apoptosis. Targeting mitochondria is a key strategy for bacteria to subvert host cells' physiology and promote infection. Helicobacter (H.) pylori targets mitochondria directly. However, mitochondrial genome (mtDNA) polymorphism (haplogroup) is not yet considered an important factor for H. pylori infection. Here, we clarified the association of mitochondrial haplogroups with H. pylori prevalence and the ability to perform damage. Seven mtDNA haplogroups were identified among 28 H. pylori-positive subjects. Haplogroup B was present at a higher frequency and haplotype D at a lower one in the H. pylori population than in that of the H. pylori-negative one. The fibroblasts carrying high-frequency haplogroup displayed a higher apoptotic rate and diminished mitochondrial respiration following H. pylori infection. mtDNA mutations were accumulated more in the H. pylori-positive population than in that of the H. pylori-negative one in old age. Among the mutations, 57% were located in RNA genes or nonsynonymous protein-coding regions in the H. pylori-positive population, while 35% were in the H. pylori-negative one. We concluded that gastric disease caused by Helicobacter virulence could be associated with haplogroups and mtDNA mutations.