Latex Beads Research Articles

Association of CD47 with Integrin Mac-1 (αMβ2, CD11b/CD18) Regulates Macrophage Responses

CD47 is a cell surface receptor, which is expressed by virtually all cells in the body, including immune cells. CD47 has originally been identified as an integrin-associated protein (IAP) and shown to associate with several integrins that belong to the β1 and β3 subfamilies. In addition, association of CD47 with a member of the β2 subfamily, integrin αLβ2, has also been reported. In neutrophils, CD47 mediates a number of integrin αvβ3-dependent functions, including adhesion, migration and phagocytosis. Surprisingly, the association of CD47 with integrin αMβ2 (Mac-1, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, has not been documented. Furthermore, while the major focus of recent studies was the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 expressed on the surface of macrophages influences the responses of these cells has not been addressed. In the present study, we demonstrated that an association of CD47 with Mac-1 regulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. In addition, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional association of CD47 with Mac-1 implied that similar to other integrins, Mac-1 might form a complex with CD47. Indeed, co-immunoprecipitation experiments using peritoneal mouse macrophages, the IC21 murine macrophage cell line and Mac-1-expressing HEK293 cells revealed that Mac-1 forms a complex with CD47. The cis interaction between Mac-1 and CD47 was also detected using the proximity ligation assay. Together, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions. Studies to determine the structural requirements for the physical interaction between Mac-1 and CD47 are in progress. DisclosuresNo relevant conflicts of interest to declare.

Rapid whole blood assay using flow cytometry for measuring phagocytic activity of chicken leukocytes

Phagocytic activity of leukocytes in whole blood was assessed as a potential immune competence trait in chickens. A flow cytometry based whole blood phagocytosis (WBP) assay was set up and evaluated using blood from chickens homozygous for four different MHC haplotypes, B12, B15, B19 and B21. Fluorescent latex beads and two serotypes of fluorescently labelled heat-killed bacteria (Salmonella Infantis and Salmonella. Typhimurium) were evaluated as phagocytic targets. In addition, the opsonophagocytic potential (OPp) of individual sera from the birds was included in a phagocytosis assay using the HD11 chicken macrophage cell line. Results showed that both serotypes of bacteria but not the latex beads were effectively phagocytosed by leukocytes in the whole blood cultures. Differences were observed in the phagocytic capacity of monocytes and thrombocyte/lymphocytes, respectively between the different MHC lines. No significant differences on the OPp of serum was identified between MHC lines. In addition, for both phagocytic activity of leukocytes and OPp of serum large variations between individuals were observed within MHC haplotypes. No significant relationships were observed between the phagocytic activity of leukocytes and serum OPp or Salmonella-specific IgY levels.In conclusion, our results suggest that the WBP assay, using a no-lyse no-wash single staining method, is a rapid and convenient method to assess phagocytic functions of different leukocyte populations.

Bruch's membrane allows unhindered passage of up to 2 μm latex beads in an in vivo porcine model

PurposeIt has been proposed that changes in the permeability of Bruch's membrane play a role in the pathogenesis of age-related macular degeneration (AMD). This paper investigates, in an in vivo porcine model, the migration of fluorescent latex beads across the Bruch's membrane after subretinal injection. MethodsForty-one healthy eyes of 33 three-month-old domestic pigs received a subretinal injection of 0.5, 1.0, 2.0, or 4.0 μm fluorescent latex beads. Between three hours and five weeks after injection evaluations were performed with fundus photographs and histology. Fluorescent beads were identified in unstained histologic sections using the rhodamine filter with the light microscope. ResultsThe fluorescent latex beads relocated from the subretinal space. Intact beads up to 2.0 μm were found in the choroid, sclera, and extrascleral space. The smaller beads were also found inside choroidal and extrascleral blood vessels. In contrast, the larger beads of 4.0 μm did not pass the Bruch's membrane. ConclusionSubretinally implanted beads up to 2.0 μm pass the Bruch's membrane intact and cross the blood-ocular barrier. The intact beads are found in the choroid, sclera and inside blood vessels. The results give reason to consider the role of subretinal clearance and passage of Bruch's membrane in the development of AMD.

A Multiplex Immunochromatographic Assay Employing Colored Latex Beads for Simultaneously Quantitative Detection of Four Nitrofuran Metabolites in Animal-Derived Food

A rapid and sensitive high-throughput latex bead immunochromatographic assay (LB-ICA) for simultaneously quantitative detection of four major nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 3-amino-2-oxazolidinone (AOZ) in animal-derived food (chicken, fish, and shrimp) was developed. Four different coating antigens were separately immobilized on nitrocellulose (NC) membrane as capture reagents; red latex beads were sprayed as constant control line. Specific monoclonal antibodies labeled with blue- or red-colored latex bead reacted with analytes in standard or sample and flowed along the strip, exhibiting visible and colorful test lines after unbound antibodies coupled with corresponding antigens on four test lines. No mutual interference was found in the simultaneous detection of the four analytes even at concentrations (100 μg L−1) higher than their respective cutoff values. The limits of detection (LODs) for SEM, AHD, AMOZ, and AOZ were 0.02–0.1 μg kg−1 in chicken, 0.02–0.15 μg kg−1 in fish, and 0.03–0.12 μg kg−1 in shrimp, respectively. Recoveries ranged from 73.5 to 109.2% at fortified concentration of LOD, 2LOD, and 4LOD, with coefficient of variation < 15%. Analysis of field animal-derived food samples by LB-ICA was in accordance with that of LC-MS/MS. Therefore, this proposed multiplex LB-ICA was a promising method for on-site screening of multiple analytes in animal-derived food.

Granulocyte macrophage-colony stimulating factor: A key modulator of renal mononuclear phagocyte plasticity

Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) play key roles in the differentiation of macrophages and dendritic cells (DCs). We examined the effect of treatment with M-CSF-containing macrophage medium or GM-CSF-containing DC medium upon the phenotype of murine bone marrow-derived macrophages and DCs. Culture of macrophages for 5 days in DC medium reduced F4/80 expression and increased CD11c expression with cells effectively stimulating T cell proliferation in a mixed lymphocyte reaction. DC medium treatment of macrophages significantly reduced phagocytosis of both apoptotic cells and latex beads and strongly induced the expression of the chemokine receptor CCR7 known to be involved in DC trafficking to lymph nodes. Lysates of obstructed murine kidneys expressed both M-CSF and GM-CSF though M-CSF expression was dominant (M-CSF:GM-CSF ratio ∼30:1). However, combination treatment with both M-CSF and GM-CSF (ratio 30:1) indicated that small amounts of GM-CSF skewed macrophages towards a DC–like phenotype. To determine whether macrophage phenotype might be modulated in vivo we tracked CD45.1+ bone marrow-derived macrophages intravenously administered to CD45.2+ mice with unilateral ureteric obstruction. Flow cytometry of enzyme dissociated kidneys harvested 3 days later indicated CD11c and MHC Class II upregulation by adoptively transferred CD45.1+ cells with CD45.1+ cells evident in draining renal lymph nodes. Our data suggests that GM-CSF modulates mononuclear phagocyte plasticity, which likely promotes resolution of injury and healing in the injured kidney.

Dielectrophoretic separation with a floating-electrode array embedded in microfabricated fluidic networks

In this study, we proposed a high-throughput separation strategy of the binary colloid mixture by dielectrophoresis (DEP) induced around large-scale bipolar electrode arrays embedded in microfabricated fluidic networks via a thorough numerical investigation. The usage of a floating electrode (FE) eliminates the need of external Ohmic connection to individual array units, therefore potentially steering the faddish design of new microdevice structures. Diffuse charge dynamics within the induced double layer at opposite ends of every FE permit a sinusoidal electric field to penetrate throughout the whole device, as long as the imposed field frequency is beyond the reciprocal resistor-capacitor time constant at the electrode/electrolyte interface. In this special device configuration, FEs interconnect multiple microchannels arranged in parallel. Pockets embedded on the sidewalls of fluidic channels help create strong field gradients at the tip of FEs and sharp pocket/channel junctions, improving the trapping performance of incoming bioparticles subjected to positive-DEP (pDEP) force, while latex beads experiencing negative-DEP (nDEP) stress are electrically squeezed to the midchannel and finally exit as a series of co-flowing thin streams with unequal translatory velocity. Taking the synergy of DEP force, induced-charge electro-osmosis, alternating-current electrothermal streaming, pressure-driven flow, and buoyancy effect into consideration, a numerical model is established to account for motion trajectories of micro-entities in full-scale three-dimensional space using the Lagrange particle track algorithm, as well as testing the feasibility of the device design in separation of the binary mixture containing yeast cells and polystyrene beads. Applying suitable voltage parameters of frequency O(1) MHz and electric field strength O(10) V/mm, highly efficient DEP separation is theoretically achievable under inlet flow velocity on the order of O(1) mm/s, where most of incoming yeasts are captured by pDEP within these five parallel branching channels, while polystyrene spheres are repelled by nDEP away from the FE array to form slim beams co-flowing into the outlet according to the calculation results. The microfluidic separation device exploiting the FE array offers great potential to build up scalable electrokinetic platforms for high-throughput on-chip sample treatment.

Analysis of phagocytic activity of macrophages of monocytic origin and Kupfer cells

The purpose of the study was to compare the phagocytic activity of macrophages of monocytic origin both without activation and under the influence of factors of the M1 and M2 phenotype. Material and methods. Peripheral blood monocytes and Kupper liver cells of male Wistar rats were obtained by gradient centrifugation. The Kupffer cells and rat monocytes were transferred to RPMI growth medium. To activate in the direction of the M1-phenotype, LPS and IFN-γ were introduced into the medium. To activate the M2 phenotype, IL 4, IL10, and IL 13 were added to the medium. The obtained macrophage cultures were stained with antibodies to CD68. To study the phagocytic activity of macrophages, the cells were placed on plates for intravital microscopy and latex beads were added to the culture medium. Results. The macrophage cultures of monocytic origin and Kupffer cells expressed CD68 at a high level, the addition of activation factors did not change the expression of the marker. 1 hour after the addition of latex particles to the culture medium, unactivated monocytic macrophages statistically significantly absorbed particles more than Kupffer cells. Activation by factors of the M1 and M2 phenotype led to an increase in the phagocytic activity of both macrophages of monocytic origin and Kupffer cells. The most activating effect on phagocytic activity was provided by induction factors of the M1 phenotype. Conclusions. For macrophages of monocytic origin and Kupffer cells, a different dynamics of phagocytic activity is characteristic. Monocytic macrophages initially have a more pronounced absorption capacity, which gradually increases during the experiment. For Kupffer cells, a sharp fluctuation of phagocytic activity is characteristic: rapid growth and rapid saturation.

Macropinocytosis-dependent endocytosis of Japanese flounder IgM+ B cells and its regulation by CD22

B cells in fish are proven to be endocytic and have a great contribution to innate immunity like phagocytosis. In this study, the endocytic capacity and the corresponding internalization pathways of IgM+ B cells in Japanese flounder (Paralichthys olivaceus) were investigated. The results showed that IgM+ B cells in peripheral blood leukocytes (PBL) and splenic leukocytes (SL) exhibited different abilities to ingest 0.5 μm and 1 μm latex beads through macropinocytosis-dependent endocytic pathway. Japanese flounder CD22 (PoCD22) co-stimulatory signals were identified to be essential for the innate immune responses in B cells. Most of IgM+ B cells and some IgM− cells were demonstrated to be PoCD22 positive. When PoCD22 was blocked by antibody, the endocytic activities and reactive oxygen species (ROS) activities of SL IgM+ B cells were significantly increased, while the endocytic and ROS activities of PBL IgM+ B cells were significant decreased. These results collectively suggest that Japanese flounder IgM+ B cells are able to employ macropinocytosis-dependent endocytic pathway, which is under the regulation of CD22.

Preparation of Template-Free Robust Yolk-Shell Gelled Particles from Controllably Evolved All-in-Water Emulsions.

A template-free all-aqueous bulk preparation of robust hollow capsules having a gelatin shell from all-in-water double emulsions is reported. The hot (>40 °C) quaternary system water/polyethylene glycol (PEG)/gelatin/alginate is shown to spontaneously form PEG-in-gelatin-in-PEG double water emulsion droplets having a multinuclear core. These droplets are stable upon cooling below the temperature at which gelatin gelled. In contrast, above the melting temperature of gelatin, multinuclear double emulsion droplets controllably evolve into stable mononuclear yolk (aqueous PEG)-shell (gelatin) capsules dispersed in the aqueous PEG continuous phase. It is demonstrated that the gelatin shell can accommodate negatively charged latex beads and be re-enforced by glutaraldehyde or silica. These capsules are also shown to encapsulate payloads, suggesting possible applications in microencapsulation, drug delivery, and synthetic biology.

Generation of Latex Films on Glassy Substrates by Evaporation Induced Self-Assembly (EISA) Process

It will be discussed in this work how it is possible to produce latex films by evaporation induced self-assembly (EISA) process. Latex beads were synthesized in a cylindrical flask without N2flow. The polymerization process of styrene happens at water as solvent and with the presence of persulfate potassium as initiator. The final product was a dispersion of spherical nanometric particles (ɸ = 0.47 ± 0.06μm) whose concentration can be changed from 0.5 to 10% (V/Vo). These dispersions were spread on glassy substrates at 50°C over a square area (~1cm2) with control of solvent evaporation. The films are transparent and they show the formation of nanowire structures by scanning electron microscopy (SEM) characterization. Such structures were associated to high affinity of latex beads particles to form cylindrical arrangements due to presence of O2during their synthesis. It has been concluded that this self-assembly structure can be very worthy for generation of functional devices like sensors, solar cells and biomaterials.

Immunomodulatory and Antioxidant Activity of Green Grass Jelly Leaf Extract (Cyclea barbata Miers.) In Vitro

Green grass jelly (Cyclea barbata Miers.) is known for its benefit to human health especially in supporting body’s immune system and wellness. This research aimed to determine immunomodulatory and antioxidant activity of green grass jelly leaf extracts in vitro. Old leaves were collected as sample then dried and ground to powder. The extraction was done with sohxletation using three different solvents, chloroform, ethyl acetate, and ethanol. The immunomodulatory activity was evaluated by treating the crude extracts at concentrations of 50, 100, and 500 mg/mL on macrophages of rat in vitro. The treated macrophage was then challenged for their phagocytic activity to latex beads. The antioxidant activity was done using 1,1-diphenil-2-picrilhydrazil (DPPH) with spectrophotometry technique. All treatments were done with three replicates. Detection of the bioactive groups of the extracts was done by Thin Layer Chromatography (TLC). The results showed that ethyl acetate extract has the highest phagocytic activity followed with chloroform extract and ethanol extract, respectively. Optimum concentration was reached at 100 mg/mL of ethyl acetat extract. The ethyl acetate extract was also the extract with the highest antioxidant activity index 7.7 followed by both extracts of chloroform and ethanol with similar index value of 6.25 and 6.3, respectively. The ethyl acetate extract contained phenolics, flavonoids, tannins, and terpenoids.

Electrospun Silver Coated Polyacrylonitrile Membranes for Water Filtration Applications.

The scarcity of drinking water and the contamination of water sources in underdeveloped countries are serious problems that require immediate low-tech and low-cost solutions. In this study, we fabricated polyacrylonitrile (PAN) porous membranes coated with silver nanoparticles (AgNP) and demonstrated their use for water filtration and water treatment applications. The membranes were prepared by electrospinning a PAN solution and treating in a hydroxylamine (NH2OH) aqueous solution to form –C(NH2)N–OH groups that were used for functionalization (Ag+ ions) of the membrane. The coordinated silver ions were then converted to silver nanoparticles. The microstructure of the membrane, water permeability, antimicrobial effect (using Escherichia coli), and particulate filtration capabilities were studied. This study verified that the membrane demonstrated a 100% reduction for Gram-negative bacteria with an effective filtration rate of 8.0 mL/cm2 min. Furthermore, the membrane was able to eliminate 60% of latex beads as small as 50 nm and over 80% of the 2 µm beads via gravity filtration. This study demonstrated that PAN–AgNP membranes can be employed as antimicrobial membranes for the filtration of water in underdeveloped countries.

Calf melanin immunomodulates RPE cell attachment to extracellular matrix protein.

It is widely accepted that RPE melanin has a protective effect against oxidative damage in RPE cells. It is possible that an additional protective characteristic of melanin is the ability to modulate RPE cell immune response. In this study, in vitro modeling was used to probe the relationship between RPE pigmentation and immune response by monitoring IL-6 expression and secretion in calf melanin pigmented ARPE-19 cells seeded onto glycated extracellular matrix as a stressor. ARPE-19 cells were left unpigmented or were pigmented with either calf melanin or latex beads, and were then seeded onto RPE-derived extracellular matrix (ECM) or tissue culture-treated plates (no ECM). ECMs were modified by glycation. IL-6 expression was measured using qPCR and IL-6 secretion was determined using an ELISA, both at 30min and 24h after seeding. MTT assay was used to quantify cell attachment to glycated matrices 30min after seeding. In unpigmented ARPE-19 cells, rate of cell attachment to substrate was monitored for 60min after seeding using a hemacytometer to count unattached cells. Additionally, cell viability was evaluated using the Neutral Red assay 24h after seeding. A significant increase in IL-6 expression was observed in calf melanin pigmented cells versus latex bead and unpigmented controls (p < 0.0001) 30min after seeding onto ECM. Twenty-four hours after seeding, a significant decrease in IL-6 expression was observed in calf melanin pigmented cells (p < 0.0001) versus controls, implicating down-regulation of the cytokine. Additionally, calf melanin pigmented cell populations showed significant increase in attachment compared to unpigmented controls on either no ECM or unmodified ECM. Pigmentation of RPE cells with calf melanin resulted in significant changes in IL-6 expression regardless of ECM modification, in vitro. These findings suggest that melanin in the RPE may participate in immune response modulation in the retina with particular regard to cell attachment to protein substrates. The results of this study further implicate the role of chemical changes to melanin in regulating inflammation in retinal disease.

Effects of bevacizumab, ranibizumab, and aflibercept on phagocytic properties in human RPE cybrids with AMD versus normal mitochondria

PurposeA critical biological function of retina pigment epithelium (RPE) cells is phagocytosis of photoreceptor outer segment (POS) disc membranes. Mitochondrial damage and dysfunction are associated with RPE cells of age-related macular degeneration (AMD) retinas. In this study, we use a transmitochondrial cybrid model to compare the phagocytic properties of RPE cells that contain AMD mitochondria versus age-matched normal mitochondria and their response to treatment with anti-vascular endothelial growth factor (VEGF) drugs: bevacizumab, ranibizumab, and aflibercept. MethodsCybrids, which are cell lines with identical nuclei but mitochondria (mt) from different subjects, are created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 5) cybrids were treated with 1 μm fluorescent latex beads (1.52 × 107 beads/mL) and either 2.09 μM of bevacizumab, 2.59 μM of ranibizumab, or 5.16 μM of aflibercept. These doses of anti-VEGF drugs are equivalent to intravitreal injections given to AMD patients with choroidal neovascularization. Flow cytometry was performed using the ImageStreamX Mark II to assess phagocytic bead-uptake. The average fold values for bead-uptake and SEM were calculated using GraphPad Prism software. ResultsNormal cybrids showed decreased bead-uptake with a fold value of 0.65 ± 0.103 (p = 0.01) after treatment with bevacizumab, 0.80 ± 0.034 (p = 0.0003) with ranibizumab, and 0.81 ± 0.053 (p = 0.007) with aflibercept compared to the untreated normal cybrids (baseline fold of 1). The bevacizumab-treated, ranibizumab-treated, and aflibercept-treated AMD cybrids had decreased bead-uptake with a fold value of 0.71 ± 0.061 (p = 0.001), 0.70 ± 0.101 (p = 0.02), and 0.74 ± 0.125 (p = 0.07), respectively, compared to the untreated AMD cybrids (baseline fold of 1). ConclusionsOur initial findings showed that when treated with bevacizumab and ranibizumab, both AMD cybrids and age-matched normal cybrids had a significant decrease in bead-uptake. A similar decrease in bead-uptake was found in normal cybrids treated with aflibercept and while the AMD values trended lower, they were not significant. This data suggests that anti-VEGF drugs can cause loss of phagocytic function.

Highly sensitivity detection of label-free H1N1 influenza A virus on electrical impedance spectroscopy using microsystems technologies

Background: H1N1 Influenza virus A is a common human pathogenic agent that caused serious respiratory illness and death over the past century. It always causes widespread pandemics whenever a new type of Influenza strain appeared in the human population and then spread person to person easily. Diagnosis of influenza is important for initiation of antiviral therapy and implementation of infection control strategies. Reverse-transcriptase polymerase chain reaction (RT-PCR) is considered the reference test for diagnosis of influenza because of its high sensitivity and specificity as well as its rapid turnaround time. However, RT-PCR is expensive and requires trained expertise and batch testing, which delays reporting. To overcome this problem, we develop a Lab-on-a-Chip (LoC) type Micro Electromechanical Systems (MEMS) scale non-invasive biosensor for detecting and differentiating infectious cells at single cell level based on their electrical properties. Methods & Materials: Finite element method (FEM) simulations were conducted in COMSOL Multi physics software to verify and optimize device design. Devices were fabricated on 4” silicon wafers with <100> orientation. Cells from four cell lines (H1N1-pdm09 and PR8-H1N1cells) were used in experiments. Cell flow through the micro fluidic channel was monitored through a video camera connected to the probe-station microscope. Impedance measurements were always taken at a low and a high frequency pair in order to obtain impedance data at two frequencies. Results: Cells from tested cell lines and latex beads were successfully detected and differentiation was demonstrated. Using linear regression, opacity (real impedance at high frequency/real impedance at low frequency) was calculated to be 0.814(latex beads), 0.200(H1N1-pdm09), 0.625(PR8-H1N1) cells respectively. R2 for data clusters were 0.777, 0.719 and 0.943 for latex beads, H1N1-pdm09 and PR8-H1N1 cells respectively. The different cell lines form clusters that are easily differentiable, thus device can be used not only to detect, but also to differentiate between the tested cell lines. Conclusion: Device exhibits potential to be developed into a LoC biosensor capable of rapid detection and analyzing different infection in blood. It requires no cell labelling, no cell immobilization or device functionalization and mean time it is very cost effective.

Glycine Protects the Liver from Reperfusion Injury following Pneumoperitoneum

Background: Experimental pneumoperitoneum induces ischemia/reperfusion injury (IRI) in the liver, most likely via Kupffer cell (KC)-dependent mechanisms. Glycine has been shown to ameliorate IRI in various animal models. Thus, this study was performed to assess the effects of glycine on the liver after pneumoperitoneum. Materials and Methods: Sprague-Dawley rats (220–250 g in weight) underwent CO₂ pneumoperitoneum (12 mm Hg) for 90 min. Some rats received i.v. glycine (1.5 mL, 300 mM) 10 min before pneumoperitoneum. Controls were given the same volume of Ringer’s solution. Transaminases, hepatic microcirculation, and phagocytosis of latex beads indexing both liver injury and KC activation were examined following pneumoperitoneum. Analysis of variance (ANOVA), plus a subsequent t test or χ² test (or Fisher’s exact test) were carried out as appropriate. Results are presented as mean ± SEM. Results: Glycine significantly decreased lactate dehydrogenase at 1 h and both aspartate aminotransferase and alanine aminotransferase at 2 h after pneumoperitoneum from 477 ± 43, 154 ± 17, and 60 ± 6 U/L in controls to 348 ± 25, 101 ± 11, and 34 ± 3 U/L, respectively (p < 0.05). In parallel, glycine significantly decreased both the rate of permanent adherence of leukocytes to the endothelium by up to 35% and the rate of phagocytosis by > 50% compared to the control group. Conclusion: Glycine decreased IRI after pneumoperitoneum, most likely via KC-dependent mechanisms.

Boosting phagocytosis and anti-inflammatory phenotype in microglia mediates neuroprotection by PPARγ agonist MDG548 in Parkinson's disease models.

Microglial phenotype and phagocytic activity are deregulated in Parkinson's disease (PD). PPARγ agonists are neuroprotective in experimental PD, but their role in regulating microglial phenotype and phagocytosis has been poorly investigated. We addressed it by using the PPARγ agonist MDG548. Murine microglial cell line MMGT12 was stimulated with LPS and/or MDG548, and their effect on phagocytosis of fluorescent microspheres or necrotic neurons was investigated by flow cytometry. Cytokines and markers of microglia phenotype, such as mannose receptor C type 1; MRC1), Ym1 and CD68 were measured by elisa and fluorescent immunohistochemistry. Levels of Beclin-1, which plays a role in microglial phagocytosis, were measured by Western blotting. In the in vivo MPTP-probenecid (MPTPp) model of PD in mice, MDG548 was tested on motor impairment, nigral neurodegeneration, microglial activation and phenotype. In LPS-stimulated microglia, MDG548 increased phagocytosis of both latex beads and necrotic cells, up-regulated the expression of MRC1, CD68 and to a lesser extent IL-10, while blocking the LPS-induced increase of TNF-α and iNOS. MDG548 also induced Beclin-1. Chronic MPTPp treatment in mice down-regulated MRC1 and TGF-β and up-regulated TNF-α and IL-1β immunoreactivity in activated CD11b-positive microglia, causing the death of nigral dopaminergic neurons. MDG548 arrested MPTPp-induced cell death, enhanced MRC1 and restored cytokine levels. This study adds a novel mechanism for PPARγ-mediated neuroprotection in PD and suggests that increasing phagocytic activity and anti-inflammatory markers may represent an effective disease-modifying approach.

In vitro phagocytosis of opsonized latex beads by HD11 cells as a method to assess the general opsonization potential of chicken serum

ABSTRACTOpsonins, an important arm of the innate immune system, are various soluble proteins, which play a critical role in destruction of invading pathogens directly or via engulfment of pathogens through the intermediate of phagocytosis. The diversity of opsonin profiles is under genetic influence and may be associated with variation in disease resistance. The aim of this study was to set up an assay to determine serum opsonophagocytic potential (OPp) for chicken sera by flow cytometry and to evaluate the assay using samples from different chicken lines. Two chicken lines selected for high and low concentrations of mannose-binding lectin, a known opsonin, in serum were used to establish the method. Furthermore, the presumed “robust” Hellevad chickens and two other commercial chicken lines (Hisex and Bovans) were tested to evaluate OPp as a parameter reflecting general immune competence. The results showed that Hellevad and Bovans chickens had higher OPp than Hisex chickens. There were no correlations between concentrations of total IgY or mannose-binding lectin and OPp. However, a strong positive correlation was observed between vaccine-induced infectious bronchitis virus titres and OPp. Moreover, inverse relationships were observed between concentrations of total serum IgM as well as natural antibody levels, and OPp. In conclusion, in vitro opsonophagocytosis assessment and determination of OPp may be of relevance when addressing general innate immunocompetence.RESEARCH HIGHLIGHTSA flow cytometry method was developed to assess poultry serum opsonophagocytosis potential.This method is based on serum-opsonin-coated polystyrene beads and HD11 cell phagocytosis.Serum samples from different commercial chicken lines were compared.Opsonophagocytic potential may be included in assay panels for general immune competence of poultry.

Myosin II Synergizes with F-Actin to Promote DNGR-1-Dependent Cross-Presentation of Dead Cell-Associated Antigens

SummaryConventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1+ cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8+ T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8+ T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design.

Microplastic and soil protists: A call for research

Microplastic is an emerging contaminant of concern in soils globally, probably gradually increasing in soil due to slow degradation. Few studies on microplastic effects on soil biota are available, and no study in a microplastic contamination context has specifically addressed soil protists. Soil protists, a phylogenetically and functionally diverse group of eukaryotic, unicellular soil organisms, are major consumers of bacteria in soils and are potentially important vehicles for the delivery of microplastics into the soil food chain. Here we build a case for focusing research on soil protists by drawing on data from previous, older studies of phagocytosis in protist taxa, which have long made use of polystyrene latex beads (microspheres). Various soil-borne taxa, including ciliates, flagellates and amoebae take up microplastic beads in the size range of a few micrometers. This included filter feeders as well as amoebae which engulf their prey. Discrimination in microplastic particle uptake depended on species, physiological state as well as particle size. Based on the results of the studies we review here, there is now a need to study microplastic effects in a pollution ecology context: this means considering a broad range of particle types under realistic conditions in the soil, and exploring longer-term effects on soil protist communities and functions.