Search Results (22)

Increased abundance of Segatella copri (S. copri) within the gut microbiota is associated with systemic inflammatory diseases, including rheumatoid arthritis. Although outer-membrane vesicles (OMVs) of Gram-negative bacteria are important players in microbiota–host communication, the effect of S. copri-derived OMVs on immune cells is unknown. Macrophages engulf and eliminate foreign material and are conditioned by environmental signals to promote either homeostasis or inflammation. Thus, we aimed to explore the impact of S. copri-OMVs on human macrophages in vitro, employing THP-1 and monocyte-derived macrophage models. The uptake of DiO-labeled S. copri-OMVs into macrophages was monitored by confocal microscopy and flow cytometry. Furthermore, the effect of S. copri and S. copri-OMVs on the phenotype and cytokine secretion of naïve (M0), pro-inflammatory (M1), and anti-inflammatory (M2) macrophages was analyzed by flow cytometry and ELISA, respectively. We show that S. copri-OMVs enter human macrophages through macropinocytosis and clathrin-dependent mechanisms. S. copri-OMVs, but not the parental bacterium, induced a dose-dependent increase in the expression of M1-related surface markers in M0 and M2 macrophages and activated the secretion of large amounts of pro-inflammatory cytokines in M1 macrophages. These results highlight an important role of S. copri-OMVs in promoting pro-inflammatory macrophage responses, which might contribute to systemic inflammatory diseases. Full article

The Staphylococcus aureus cell wall protein serine rich adhesin for platelets (SraP) belongs to a large surface glycoprotein family of adhesins. Here, we provide experimental evidence that SraP mediates macrophage functions in a human monocyte-derived macrophage model via its N-terminal L-lectin module (LLM) in the ligand binding region. Our flow cytometry data demonstrated that macrophages infected by the LLM deletion strain profoundly impacted apoptosis, reducing the percentage of apoptotic cells by approximately 50%, whereas LLM overexpression significantly increased the percentage of early-stage apoptotic cells (p < 0.001). LLM deletion significantly enhanced phagocytosis by macrophages by increasing the number of engulfed bacteria, resulting in a significant increase in bacterial killing and leading to a notable decrease in bacterial survival within macrophages (p < 0.001). Furthermore, LLM modulated the ability of S. aureus to elicit inflammatory responses. The LLM deletion strain dampened the expression of proinflammatory factors but increased the expression of anti-inflammatory cytokines, such as IL10. Our evidence suggests that SraP likely plays a dual role in S. aureus pathogenesis, by acting as a virulence factor involved in bacterial adhesion and invasion and by mediating macrophage functions. Our future work will focus on the identification of small molecule inhibitors of LLM using molecular docking-based in silico screening and in vivo validation. Developing LLM inhibitors, alone or in combination with conventional antibiotics, may represent a novel strategy for combating S. aureus infections. Full article

In healthy conditions, blood was considered a sterile environment until the development of new analytical approaches that allowed for the detection of circulating bacterial ribosomal DNA. Currently, debate exists on the origin of the blood microbiota. According to advanced research using dark field microscopy, fluorescent in situ hybridization, flow cytometry, and electron microscopy, so-called microbiota have been detected in the blood. Conversely, others have reported no evidence of a common blood microbiota. Then, it was hypothesized that blood microbiota may derive from distant sites, e.g., the gut or external contamination of blood samples. Alteration of the blood microbiota’s equilibrium may lead to dysbiosis and, in certain cases, disease. Cardiovascular, respiratory, hepatic, kidney, neoplastic, and immune diseases have been associated with the presence of Gram-positive and Gram-negative bacteria and/or their products in the blood. For instance, lipopolysaccharides (LPSs) and endotoxins may contribute to tissue damage, fueling chronic inflammation. Blood bacteria can interact with immune cells, especially with monocytes that engulf microorganisms and T lymphocytes via spontaneous binding to their membranes. Moreover, LPSs, extracellular vesicles, and outer membrane vesicles interact with red blood cells and immune cells, reaching distant organs. This review aims to describe the composition of blood microbiota in healthy individuals and those with disease conditions. Furthermore, special emphasis is placed on the interaction of blood microbiota with host cells to better understand disease mechanisms. Full article

Insufficient drug accumulation in tumors is still a major concern for using cancer nanotherapeutics. Here, the neutrophil-based delivery of three nanoparticle types—liposomes, PLGA, and magnetite nanoparticles—was assessed both in vitro and in vivo. Confocal microscopy and a flow cytometry analysis demonstrated that all the studied nanoparticles interacted with neutrophils from the peripheral blood of mice with 4T1 mammary adenocarcinoma without a significant impact on neutrophil viability or activation state. Intravital microscopy of the tumor microenvironment showed that the neutrophils did not engulf the liposomes after intravenous administration, but facilitated nanoparticle extravasation in tumors through micro- and macroleakages. PLGA accumulated along the vessel walls in the form of local clusters. Later, PLGA nanoparticle-loaded neutrophils were found to cross the vascular barrier and migrate towards the tumor core. The magnetite nanoparticles extravasated in tumors both via spontaneous macroleakages and on neutrophils. Overall, the specific type of nanoparticles largely determined their behavior in blood vessels and their neutrophil-mediated delivery to the tumor. Since neutrophils are the first to migrate to the site of inflammation, they can increase nanodrug delivery effectiveness for nanomedicine application. Full article

Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy. Autophagy is implicated in human pathophysiology, such as neurodegeneration, cancer, and immune disorders. Moreover, autophagy impacts urological diseases, such as interstitial cystitis /bladder pain syndrome (IC/BPS), ketamine-induced ulcerative cystitis (KIC), chemotherapy-induced cystitis (CIC), radiation cystitis (RC), erectile dysfunction (ED), bladder outlet obstruction (BOO), prostate cancer, bladder cancer, renal cancer, testicular cancer, and penile cancer. Autophagy plays a dual role in the management of urologic diseases, and the identification of potential biomarkers associated with autophagy is a crucial step towards a deeper understanding of its role in these diseases. Methods for monitoring autophagy include TEM, Western blot, immunofluorescence, flow cytometry, and genetic tools. Autophagosome and autolysosome structures are discerned via TEM. Western blot, immunofluorescence, northern blot, and RT-PCR assess protein/mRNA levels. Luciferase assay tracks flux; GFP-LC3 transgenic mice aid study. Knockdown methods (miRNA and RNAi) offer insights. This article extensively examines autophagy’s molecular mechanism, pharmacological regulation, and therapeutic application involvement in urological diseases. Full article

Membranous CD14 is crucial in the phagocytic activity of neutrophils. However, the role of CD14(+) microparticles (MPs) derived from apoptotic neutrophils (apo-MP) during the phagocytic process is not clear. All trans-retinoic acid (ATRA) induces acute promyelocytic leukemic NB4 cells along granulocytic differentiation. In this study, we investigated the role of CD14(+)apo-MP in the cell–cell interaction during the phagocytic process of apoptotic cells by viable ATRA-NB4 cells. We firstly demonstrate that CD14 expression and phagocytic activity of NB4 cells were upregulated simultaneously after ATRA treatment in a time-dependent manner, and both were significantly enhanced via concurrent lipopolysaccharide treatment. The phagocytic activity of ATRA-NB4 cells and lipopolysaccharide-treated ATRA-NB4 cells were both significantly attenuated by pre-treating cells with an antibody specific to either CD14 or TLR4. Further flow cytometric analysis demonstrates that apoptotic ATRA-NB4 cells release CD14(+)apo-MP in an idarubicin dosage-dependent manner. Both CD14 expression and the phagocytic activity of viable ATRA-NB4 cells were significantly enhanced after incubation with apo-MP harvested from apoptotic ATRA-NB4 cells, and the apo-MP-enhanced phagocytic activity was significantly attenuated by pre-treating apo-MP with an anti-CD14 antibody before incubation with viable cells. We conclude that CD14(+)apo-MP derived from apoptotic ATRA-NB4 cells promotes the phagocytic activity of viable ATRA-NB4 cells in engulfing apoptotic cells. Full article

The Taylor–Couette reactor (TCR) is becoming an increasingly significant topic in chemical industry. This study investigates the micromixing performance of a ribbed TCR with axial flow in the Villermaux–Dushman reaction system. The local micromixing mechanism of the ribbed TCR was analyzed, and the volume-averaged energy dissipation rate was calculated using CFD. The effects of operating parameters and rib structural parameters on micromixing performance were investigated. The results show that the introduction of ribs eliminates the high shear region between the vortex pairs, resulting in the strong micromixing region being situated on the inner and outer cylinder wall surfaces and the ribbed surface region. Smaller rib spacing, larger rib width, and rib height can strengthen micromixing and result in a smaller segregation index. Micromixing times of ribbed TCRs were calculated using the incorporation model, t_m, in the range of 2.0 × 10⁻⁵ to 8.0 × 10⁻³. The results show that ribbed TCRs require a lower energy consumption to achieve a lower t_m than other rotating reactors. A correlation equation between t_m and five parameters was developed, with a correlation coefficient of 0.951. The accuracy of the volume-averaged energy dissipation rate obtained via CFD was verified through experimental analysis. The correlation between the micromixing time and the volume-averaged energy dissipation rate was established in a form that satisfies Kolmogorov’s turbulence theory for t_m. To convert the volume-averaged energy dissipation rate into a local energy dissipation rate, a factor ϕ was introduced and solved using the engulfing diffusion model. This study provides insights into the design and optimization of ribbed TCRs. Full article

Distribution of inclusions plays an essential role in inducing intracrystalline ferrite, and the migration behavior of inclusions during solidification has a significant influence on their distribution. The solidification process of DH36 (ASTMA36) steel and the migration behavior of inclusions at the solidification front were observed in situ using high-temperature laser confocal microscopy. The annexation, rejection, and drift behavior of inclusions in the solid–liquid two-phase region were analyzed, providing a theoretical basis for regulating the distribution of inclusions. Analysis of inclusion trajectories showed that the velocity of inclusions decreases significantly as they near the solidification front. Further study of the force on inclusions at the solidification frontier shows three situations: attraction, repulsion, and no influence. Additionally, a pulsed magnetic field was applied during the solidification process. The original dendritic growth mode changed to that of equiaxed crystals. The compelling attraction distance for inclusion particles with a diameter of 6 μm at the solidification interface front increased from 46 μm to 89 μm, i.e., the effective length for the solidification front engulfing inclusions can be increased by controlling the flow of molten steel. Full article

IgE-mediated allergic reaction occurs in response to harmless environmental compounds, such as tree and grass pollen, fragments of household microorganisms, etc. To date, the only way to treat IgE-mediated allergy is allergen-specific immunotherapy (ASIT), which consists of a prolonged subcutaneous administration of allergen extracts or recombinant proteins. The long duration of the treatment, the cost and the risk of life-threatening adverse reactions are the main limiting factors for ASIT. The aim of this work was to develop allergen proteins encapsulated in chitosan-based microparticles that can be safely administered at high doses and in a rash protocol. The egg white allergen, Gal d 1 protein, was used as a model antigen. The protein was packed into core–shell type microparticles (MPs), in which the core was formed with succinyl chitosan conjugated to Gal d 1, subsequently coated with a shell formed by quaternized chitosan. The obtained core–shell MPs containing Gal d 1 in the core (Gal-MPs) were non-toxic to macrophage and fibroblast cell lines. At the same time, Gal-MPs were quickly engulfed by bone marrow-derived dendritic cells or RAW264.7 macrophage cells, as was visualized using flow cytometry and confocal microscopy. Encapsulated Gal d 1 was not recognized by Gal d 1-specific IgE in ELISA. Female BALB/c mice were immunized with Gal-MPs subcutaneously three times a week for 2 weeks. Immunization of mice resulted in IgG titers 1250 ± 200 without IgE production. Allergy in control and vaccinated mice was induced by low-dose Gal d 1 injections in the withers of mice. IgE was induced in control-sensitized but not in the vaccinated mice. Thus, preventive vaccination with the encapsulated allergens is safe and rapid; it significantly reduces the risk of IgE production induced by respiratory and oral allergens. Full article

To understand the influence of indigestible particles like particulate matter 2.5 (PM2.5) on macrophages, we examined the time course of the series phagocytosis of indigestible 2 μm polystyrene spheres (PS). Five kinds of antigens were used as samples for phagocytosis; Zymosan, non-coated 2 $\mathsf{\mu }$m PS, bovine serum albumin (BSA)-coated PS (BSA-PS), IgG-coated PS (IgG-PS), and IgG-BSA-coated PS (IgG/BSA-PS). To keep the surrounding concentration of antigens against single macrophages constant, antigens flowed at a continuous rate of $0.55$ μm/s within a culture dish as a free-flow measurement assay (on-chip free-flow method). The interval of series phagocytosis for IgG/BSA-PS was the shortest among five samples; it was six times faster than Zymosan in terms of engulfment frequency, and up to 50 particles were engulfed within two hours, maintaining constant intervals until reaching the maximum number. The rate of increase in the total number of phagocytozed IgG/BSA-PS over time was constant, at $1.5$ particles/min, in series phagocytosis with a 33-cell population, indicating that the phagocytosis rate constant remained constant independent of the number of phagocytoses. Reaction model fitting of the results showed that IgG/BSA-PS had the highest efficiency in terms of the phagocytosis rate constant, 2.3 × ${10}^{-2}$ particles/min, whereas those of IgG-PS, BSA-PS, PS, and Zymosan were 1.4 × ${10}^{-2}$, 1.1 × ${10}^{-2}$, 4.2 × ${10}^{-3}$, and 3.6 × ${10}^{-3}$ particles/min, respectively. One-by-one feeding of IgG/BSA-PS with optical tweezers was examined to confirm the phagocytosis intervals, and we found that the intervals remained constant until several times before the maximum number of antigens for engulfment, also indicating no change in the phagocytosis rate constant regardless of the history of former phagocytosis and phagocytosis number. Simultaneous phagocytosis of two IgG-BSA-decorated microneedle engulfments also showed that the initiation and progress of two simultaneous engulfments on the two different places on a cell were independent and had the same elongation velocity. Therefore, each phagocytosis of indigestible antigens does not affect both in series or in simultaneous subsequent phagocytosis until reaching the maximum capacity of the phagocytosis number. The results suggest (1) no change in the phagocytosis rate constant regardless of the history of phagocytosis numbers and attachment timing and positions, and (2) IgG-BSA decoration of indigestible microparticles in blood accelerates their engulfment faster, resulting in a severe shortage of macrophages within the shortest time. Full article

Under quasi-steady particle pushing conditions in an alloy, fresh liquid has to flow to the gap separating a particle and an advancing solid–liquid interface of a crystal to feed the volume change associated with the liquid–solid phase transformation. In the meantime, solute rejected by the growing crystal has to diffuse out of the gap against the physical feeding flow. An inequality equation was derived to estimate the pushing-to-engulfment transition (PET) velocity of the crystal under which the particle is pushed by the growing crystal. Experiments were performed in an Al-4.5 wt.%Cu-2 wt.% TiB₂ composite under isothermal coarsening conditions. TiB₂ particles were indeed engulfed by the growing aluminum dendrites as predicted using the inequality equation. Predictions of the inequality equation also agreed reasonably well with literature data from the solidification of distilled water containing particles obtained under minimal convection conditions. The inequality equation suggests that the PET velocity is much smaller in a binary alloy than that in a pure material. Without the influence of fluid flow or other factors that put a particle in motion in the liquid, the particle should be engulfed by the growing crystal in alloys solidified under normal cooling rates associated with convectional casting conditions. Full article

A new model of particle entrapment during continuous casting of steel is presented, which includes the effects of multiphase flow from argon gas injection and thermal buoyancy from superheat in the strand. The model simulates three different capture mechanisms, including capture by solidified hooks at the meniscus, entrapment between dendrites, and engulfment by the surrounding of large particles. The fluid flow and bubble capture results are validated with plant measurements, including nail board dipping tests and ultrasonic tests, respectively, and good agreement is seen. Results suggest that the superheat has a negligible effect on the flow in the mold region. However, higher (30 K) superheat causes a more complex flow in the lower strand by creating multiple recirculation zones due to the thermal buoyancy effects. This causes less penetration deep into the strand, which leads to fewer and shallower particle captures. Lower (10 K) superheat may enable significant top surface freezing, leading to very large internal defect clusters. Lower superheat also leads to deeper meniscus hooks, which sometimes (0.003%) capture large (1 mm) bubbles. Capture bands occur near the transition line from vertical to curved, due to the downward fluid velocity balancing the particle terminal velocity, enabling capture in the relative stagnation region beneath the longitudinal recirculation zone. These findings agree with plant observations. Full article

Silicified wood occurs abundantly in Middle Miocene flows and sedimentary interbeds of the Columbia River Basalt Group (CRBG) in central Washington State, USA. These fossil localities are well-dated based on radiometric ages determined for the host lava. Paleoenvironments include wood transported by lahars (Ginkgo Petrified Forest State Park), fluvial and palludal environments (Saddle Mountain and Yakima Canyon fossil localities), and standing forests engulfed by advancing lava (Yakima Ridge fossil forest). At all of these localities, the mineralogy of fossil wood is diverse, with silica minerals that include opal-A, opal-CT, chalcedony, and macrocrystalline quartz. Some specimens are composed of only a single form of silica; more commonly, specimens contain multiple phases. Opal-A and Opal-CT often coexist. Some woods are mineralized only with chalcedony; however, chalcedony and macrocrystalline quartz are common as minor constituents in opal wood. In these specimens, crystalline silica filling fractures, rot pockets, and cell lumen may occur. These occurrences are evidence that silicification occurred as a sequential process, where changes in the geochemical environment or anatomical structures affected the precipitation of silica. Fossilization typically began with precipitation of amorphous silica within cell walls, leaving cell lumen and conductive vessels open. Diagenetic transformation of opal-A to opal-CT in fossil wood has long been a widely accepted hypothesis; however, in opaline CRBG specimens, the two silica polymorphs usually appear to have formed independently, e.g., woods in which cell walls are mineralized with opal-A but in which lumen contain opal-CT. Similarly, opal-CT has been inferred to sometimes transform to chalcedony; however, in CRBG, these mixed assemblages commonly resulted from multiple mineralization episodes. Full article

The T-shaped micro-junction is among the most used geometry in microfluidic applications, and many design modifications of the channel walls have been proposed to enhance mixing. In this work, we investigate through numerical simulations the introduction of one pair of small rectangular cavities in the lateral walls of the mixing channel just downstream of the confluence region. The aim is to preserve the simple geometry that has contributed to spread the practical use of the T-shaped micro-junction while suggesting a modification that should, in principle, work jointly with the vortical structures present in the mixing channel, further enhancing their efficiency in mixing without significant additional pressure drops. The performance is analyzed in the different flow regimes occurring by increasing the Reynolds number. The cavities are effective in the two highly-mixed flow regimes, viz., the steady engulfment and the periodic asymmetric regimes. This presence does not interfere with the formation of the vortical structures that promote mixing by convection in these two regimes, but it further enhances the mixing of the inlet streams in the near-wall region of the mixing channel without any additional cost, leading to better performance than the classical configuration. Full article

Carnosine (β-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide has well-known antioxidant, anti-inflammatory, and anti-aggregation activities, and it may be useful for treatment of neurodegenerative disorders such as Alzheimer’s disease (AD). In this disease, peripheral infiltrating macrophages play a substantial role in the clearance of amyloid beta (Aβ) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aβ have been reported. The effects of carnosine on macrophages and oxidative stress associated with AD are consequently of substantial interest for drug discovery in this field. In the present work, a model of stress induced by Aβ1-42 oligomers was investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR. These assays were used to assess the ability of carnosine to protect macrophage cells, modulate oxidative stress, and profile the expression of genes related to inflammation and pro- and antioxidant systems. We found that pre-treatment of RAW 264.7 macrophages with carnosine counteracted cell death and apoptosis induced by Aβ1-42 oligomers by decreasing oxidative stress as measured by levels of intracellular nitric oxide (NO)/reactive oxygen species (ROS) and production of peroxynitrite. This protective activity of carnosine was not mediated by modulation of the canonical inflammatory pathway but instead can be explained by the well-known antioxidant and free-radical scavenging activities of carnosine, enhanced macrophage phagocytic activity, and the rescue of fractalkine receptor CX3CR1. These new findings obtained with macrophages challenged with Aβ1-42 oligomers, along with the well-known multimodal mechanism of action of carnosine in vitro and in vivo, substantiate the therapeutic potential of this dipeptide in the context of AD pathology. Full article