Latex Beads Research Articles

Phagocytic activity and proinflammatory activation potential of smooth muscle cells of the Tunica intima of human aorta under experimental conditions

The aorta is the largest blood vessel in the body, which reaches a diameter of about 3 cm and is responsible for transporting oxygen-enriched arterial blood from the heart to tissues and organs. The aortic wall consists of three layers: the inner Tunica intima, the middle Tunica media and the outer Tunica adventitia. The layers of the aortic wall have a diverse cellular composition and include smooth muscle cells (SMCs), fibroblasts, endothelial cells, etc. Functional disorders in the cells of the intima of the human aorta can lead to various cardiovascular diseases (СVD), such as aneurysm and, as a result, dissection or rupture of the thoracic aorta. The cellular and molecular mechanisms of CVD development remain not fully understood, therefore, the study of the functional characteristics of various cell populations that make up the human aorta is an urgent task today. The aim is to evaluate the inflammatory response formed by cells that are part of the Tunica intima of the human aorta during phagocytosis of latex particles and internalization of low-density lipoproteins (LDL) to study their role in the development of aneurysms. The experiments were performed on smooth muscle cells (SMCs) isolated from the intima of the human aorta in patients with aneurysm. Phagocytic activity was studied by adding latex beads to the SMCs of Tunica intima, the ability to internalize LDL was evaluated using the BDP 630/650 dye and a biochemical method, the assessment of the ability to pro- and anti-inflammatory activation was studied using enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that the absorption of latex beads and LDL stimulates the activation of interleukin secretion by smooth muscle cells that are part of the Tunica intima of the aorta, namely the proinflammatory cytokines IL-6 and IL-8. This fact may indicate that in the conditions of the body, the human aortic intima SMCs phenotype may switch from contractile to secretory or macrophage-like, which indicates the participation of this phenotypic cell transition in the process of aneurysm development.

A rapid point-of-care population-scale dipstick assay to identify and differentiate SARS-CoV-2 variants in COVID-19-positive patients.

Delta and Omicron variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are remarkably contagious, and have been recognized as variants of concern (VOC). The acquisition of spontaneous substitutions or insertion-deletion mutations (indels) in the spike protein-encoding gene substantially increases the binding affinity of the receptor binding domain (RBD)-hACE2 complex and upsurges the transmission of both variants. In this study, we analyzed thousands of genome sequences from 30 distinct SARS-CoV-2 variants, focusing on the unique nucleic acid signatures in the spike gene specific to the Delta and Omicron variants. Using these variant-specific sequences, we synthesized a range of oligonucleotides and optimized a multiplex PCR (mPCR) assay capable of accurately identifying and differentiating between the Delta and Omicron variants. Building on this mPCR assay, we developed a dipstick format by incorporating a tag linker sequence at the 5' end of the forward primer and adding biotin to the 3' end of the oligonucleotides, enhancing the assay's usability and accessibility. Streptavidin-coated latex beads and the dipstick imprinted with a probe for the tag linker sequence in the test strips were used for the detection assay. Our dipstick-based assay, developed as a rapid point-of-care test for identifying and differentiating SARS-CoV-2 variants has the potential to be used in low-resource settings and scaled up to the population level.

Morphological and functional characterization of circulating hemocytes in Tribolium castaneum larvae.

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye-staining properties were used to characterize hemocyte types from T.castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2h), then increased over time (at 4 and 6h) and eventually returned to the naive state by 24h post-injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T.castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph invivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes invitro, forming capsules to encapsulate and melanize nickel-nitrilotriacetic acid (Ni-NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T.castaneum larvae, offering valuable insights into cell-mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T.castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T.castaneum hemocytes in combating pathogens.

Microglial Melatonin Receptor 1 Degrades Pathological Alpha-Synuclein Through Activating LC3-Associated Phagocytosis InVitro.

Parkinson's disease (PD) is characterized by the formation of Lewy bodies (LBs), primarily constituted of α-synuclein (α-Syn). Microglial cells exhibit specific reactivity toward misfolded proteins such as α-Syn. However, the exact clearance mechanism and related molecular targets remain elusive. BV2 cells, primary microglia from wild-type and MT1 knockout mice, and primary cortical neurons were utilized as experimental models. The study investigated relevant mechanisms by modulating microglial MT1 expression through small RNA interference (RNAi) and lentiviral overexpression techniques. Furthermore, pathological aggregation of α-Syn was induced using pre-formed fibrils (PFF) α-Syn. Co-immunoprecipitation, immunofluorescence, Western blot (WB), and quantitative real-time PCR were used to elucidate the mechanisms of molecular regulation. In this study, we elucidated the regulatory role of the melatonin receptor 1 (MT1) in the microglial phagocytic process. Following MT1 knockout, the ability of microglial cells to engulf latex beads and zymosan particles decreased, subsequently affecting the phagocytic degradation of fibrillar α-Syn by microglial cells. Furthermore, the loss of MT1 receptors in microglial cells exacerbates the aggregation of α-Syn in neurons induced by pre-formed fibrils (PFF) α-Syn. Mechanistically, MT1 influences the phagocytic function of microglial cells by regulating the Rubicon-dependent LC3-associated phagocytosis (LAP) pathway. Taken together, the results suggest the neuroprotective function of microglial cells in clearing α-Syn through MT1-mediated LAP, highlighting the potential key role of MT1 in pathogenic mechanisms associated with α-Syn.

Functional features of smooth muscle cells of the human aortic wall and their role in the pathogenesis of aneurysms

BACKGROUND: Thoracic aortic aneurysm is a chronic disease characterized by localized dilation of the vessel, including the ascending, arch and descending parts of the aorta. Aneurysm is the most dangerous disease because aortic dissection or rupture can lead to sudden death. More than 150,000 people worldwide die from aortic aneurysms every year. Despite the prevalence of this disease, the cellular mechanisms of its development remain not fully understood. AIM: evaluation of phagocytic activity and the ability to proinflammatory activation of smooth muscle cells isolated from Tunica intima and Tunica media of the thoracic part of the human aorta in patients with aneurysm. METHODS: The experiments were performed on a culture of primary smooth muscle cells (SMC) isolated from aneurysm patients, linear cultures (mesenchymal stem cells ASC52telo, fibroblasts 977hTERT, THP-1, CLTH-CL05008 and EA.hy926) and SMC isolated from healthy donors were used as controls. Phagocytic activity was studied by adding latex beads to the studied cells, the ability to internalize LDL was evaluated using the dye BDP 630/650 (Lumiprobe, Russia) and a biochemical method, the assessment of the ability to pro- and anti-inflammatory activation was studied using enzyme-linked immunosorbent assay (ELISA), during which the amount of secreted cytokines was measured: IL-8, IL-6 and IL-10. RESULTS: It has been shown that the studied cells have high phagocytic activity and the ability to internalize LDL. Thus, primary SMC from Tunica media have greater phagocytic activity than SMC obtained from healthy donors (p 0.001). In addition, IL-6 secretion after incubation with latex beads was significantly higher in SMC from Tunica media, compared with SMC from healthy donors (p 0.001). Also, IL-6 secretion after incubation with LDL was higher in SMC from Tunica intima, compared with SMC from healthy donors (p 0.001). CONCLUSION: Thus, the absorption of latex beads and LDL stimulates the secretion of proinflammatory IL-6 by primary SMC from Tunica intima and SMC from Tunica media, which are part of the aortic wall of patients with aneurysm.

Glucomannan is a promising isoniazid's enhancer that inducing macrophage phagocytosis.

Isoniazid (INH) is a frontline antituberculosis agent effective against Mycobacterium tuberculosis (Mtb), but the increasing challenge of avoiding multidrug-resistant tuberculosis, including INH resistance, necessitates innovative approaches. This study focused on enhancing macrophage phagocytosis to overcome INH resistance. Glucomannan, an immunomodulatory polysaccharide, emerged as a potential macrophage activator. Our objective was to characterize the glucomannan-INH mixture and assess its impact on INH efficacy and macrophage activity. Detailed examination of the glucomannan from Amorphophallus muelleri (0.05%-0.2%) was performed in several methods. INH sensitivity tests were carried out with the Mtb strain H37RV on Löwenstein-Jensen medium. Murine macrophage (RAW264.7) viability and activity were evaluated through MTT and latex bead phagocytosis assays. Ultraviolet-wavelength spectrophotometry was used to analyze chemical structure changes. Glucomannan (0.05%-0.2%) significantly enhanced murine macrophage viability and activity. When glucomannan was combined with INH, the IC50 value was greater compared to INH only. Phagocytosis assays revealed heightened macrophage activity in the presence of 0.05% and 0.1% glucomannan. Importantly, glucomannan did not compromise INH efficacy or alter its chemical structure. This study underscores the potential of glucomannan, particularly with a lower molecular weight, as a promising enhancer of INH, boosting macrophage phagocytosis against INH-resistant Mtb. These findings challenge the assumptions about the impact of glucomannan on drug absorption and prompt potential reevaluation. While specific receptors for glucomannan in macrophage phagocytosis require further exploration, the complement receptors are proposed to be potential mediators.

Proteomic insights into myopia development: Differential protein expression and the role of calcium signaling in form deprivation myopia in Guinea pigs

This study aims to explore the changes in the vitreous proteomics of form deprivation myopia (FDM) in guinea pigs, in order to reveal the molecular mechanisms involved in the onset and development of myopia. The myopia model in guinea pigs was successfully established by covering one eye of the guinea pigs with a latex bead sac for 4 weeks. This study used 4D data-independent acquisition proteomics technology to analyze vitreous body samples from both the FDM group and the control group. The goal of the proteomics analysis was to identify differences in protein expression within the vitreous body of FDM guinea pigs. Myopia was successfully induced in the FDM group after 4 weeks of modeling. A total of 6298 proteins were identified, among which 348 were differentially expressed proteins (DEPs), with 81 upregulated and 267 downregulated. These DEPs were subjected to in-depth bioinformatics analyses, including Gene Ontology, the Eukaryotic Orthologous Groups, and the Kyoto Encyclopedia of Genes and Genomes. These analyses revealed significant involvement in cellular processes, metabolic pathways, biological regulation, cytoskeletal organization, and cell movement. Our results indicate that calcium signaling plays a critical role in mediating eye changes associated with form deprivation, which may bear similarities to mechanisms observed in neurodegenerative diseases. A total of 348 DEPs related to the development and progression of myopia were identified. These changes involve key biological processes, including protein degradation, cell adhesion, and transport, especially alterations in calcium signaling pathways. Stromal interaction molecule 1 (STIM1) is an important biological marker of FDM, which was confirmed by Western blot, immunohistochemistry and ELISA. Our study found clear differences in the expression of proteins in the vitreous during the development of myopic guinea pigs, especially those related to calcium signaling pathway. Our study offers new insights into the pathogenesis of myopia, particularly changes related to protein metabolism pathways.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is acellularreceptor for delta inulin adjuvant.

Delta inulin, or Advax, is a polysaccharide vaccine adjuvant that significantly enhances vaccine-mediated immune responses against multiple pathogens and was recently licensed for use in the coronavirus disease 2019 (COVID-19) vaccine SpikoGen. Although Advax has proven effective as an immune adjuvant, its specific binding targets have not been characterized. In this report, we identify a cellular receptor for Advax recognition. In vitro uptake of Advax particles by macrophage cell lines was substantially greater than that of latex beads of comparable size, suggesting an active uptake mechanism by phagocytic cells. Using a lectin array, Advax particles were recognized by lectins specific for various carbohydrate structures including mannosyl, N-acetylgalactosamine and galactose moieties. Expression in nonphagocytic cells of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a C-type lectin receptor, resulted in enhanced uptake of fluorescent Advax particles compared with mock-transfected cells. Advax uptake was reduced with the addition of ethylenediaminetetraacetic acid and mannan to cells, which are known inhibitors of DC-SIGN function. Finally, a specific blockade of DC-SIGN using a neutralizing antibody abrogated Advax uptake in DC-SIGN-expressing cells. Together, these results identify DC-SIGN as a putative receptor for Advax. Given the known immunomodulatory role of DC-SIGN, the findings described here have implications for the use of Advax adjuvants in humans and inform future mechanistic studies.

Optical polarization studies of latex beads in aqueous solution: An analog for radar scattering in icy regolith

This study presents low phase angle 0°–5° measurements of polarimetric properties of icy planetary regolith analog materials acquired using the custom-built Multi-Axis- Goniometer-Instrument (MAGI). We present same sense (SC), and opposite sense (OC) backscatter circular polarization coefficients, circular polarization ratio (CPR), and degree linear polarization (DLP) of spherical latex (non-dye) beads of varying sizes and volume concentrations (v/v) in aqueous solutions (λ=0.8μm) in water. We also present measurements of alumina powder in air at λ=1.064μm. Measurements showed that at a low incidence angle (i=0°), backscatter is dominated by surface specular single-bounce scattering, that hides other scattering processes. At high (i=15°) incidence angle, surface single-bounce surface scattering becomes negligible, allowing for the detection of diffuse, dihedral (multiple bounces) scattering. We find that classical Mie alumina particles (2.1μm,4.0μm) enhance subsurface scattering due to a larger void space relative to larger Mie particles (30μm), that cause the radar signal to scatter forward off small imperfections, maintaining the polarization properties of the signal and generating high >1CPR. Latex beads, representing impurities, demonstrate the impact of isotropic and anisotropic scattering on radar signatures. This study also found that the scattering medium’s anisotropy correlates to the size of the beads, while the void space of the medium inversely correlates with the bead size and the volume concentration (v/v) of the beads. Rayleigh beads, due to isotropic scattering from the reduced scattering cross-section and higher transparency relative to larger impurities, generate subsurface single bounce scattering from the sample platform, producing OC≫SC and a low (<0.5)CPR across all v/v. Rayleigh impurities in transparent water-ice simulate single bounce scattering from underlying layers. Conversely, classical and large Mie beads generate anisotropic scattering that intensifies scattering in the forward direction with high CPR, inversely proportional to the volume concentration. This study aids in interpreting radar observations of icy bodies in the solar system, providing insights into the interplay between radar waves and icy regolith compositions.

Prediction models for the flux decay profile and initial flux of microfiltration for therapeutic proteins.

Microfiltration (MF) is an essential step during biopharmaceutical manufacturing. However, unexpected flux decay can occur. Although the flux decay profile and initial flux are important factors determining MF filterability, predicting them accurately is challenging, as the root cause of unexpected flux decay remains elusive. In this study, the methodology for developing a prediction model of flux decay profiles was established. First, the filtration profiles of different monodisperse polystyrene latex and silica beads of various sizes were evaluated. These results revealed that the size and surface electrostatic properties of the beads affect the flux decay profile. Taking the size and surface electrostatic properties of protein aggregates into account, we constructed a predictive model using model bead filtration profiles. We showed that this methodology was applicable to two different MF filters to predict the flux decay profile of therapeutic proteins. Because our proposed prediction model is based on normalized flux, the initial flux is required to predict the overall filtration profile. Then, we applied the Hagen-Poiseuille equation using sample viscosity values to estimate the initial flux. The developed prediction models can be used for effective MF scale-up assessment during the early stages of process development.

Cell-particles interaction – selective uptake and transport of microdiamonds

Diamond particles have recently emerged as novel agents in cellular studies because of their superb biocompatibility. Their unique characteristics, including small size and the presence of fluorescent color centers, stimulate many important applications. However, the mechanism of interaction between cells and diamond particles—uptake, transport, and final localization within cells—is not yet fully understood. Herein, we show a novel, to the best of our knowledge, cell behavior wherein cells actively target and uptake diamond particles rather than latex beads from their surroundings, followed by their active transport within cells. Furthermore, we demonstrate that myosin-X is involved in cell-particle interaction, while myosin-II does not participate in particle uptake and transport. These results can have important implications for drug delivery and improve sensing methods that use diamond particles.

Detection of α-Thrombin with Platelet Glycoprotein Ibα (GP1bα) for the Development of a Coagulation Marker.

The detection of prothrombotic markers is crucial for understanding thromboembolism and assessing the effectiveness of anticoagulant drugs. α-Thrombin is a marker that plays a critical role in the coagulation cascade process. However, the detection of this enzymatic molecule was hindered by the absence of an efficient modality in the clinical environment. Previously, we reported that one α-thrombin interacts with two α-chains of glycoprotein Ib (GPIbα), i.e., multivalent protein binding (MPB), using bioresponsive hydrogel nanoparticles (nanogels) and optical microscopy. In this study, we demonstrated that GPIbα-mediated platforms led to the highly sensitive and quantitative detection of α-thrombin in various diagnostic systems. Initially, a bioresponsive nanogel-based surface plasmon resonance (nSPR) assay was developed that responds to the MPB of α-thrombin to GPIbα. The use of GPIbα for the detection of α-thrombin was further validated using the enzyme-linked immunosorbent assay, which is a gold-standard protein detection technique. Additionally, GPIbα-functionalized latex beads were developed to perform latex agglutination (LA) assays, which are widely used with hospital diagnostic instruments. Notably, the nSPR and LA assays exhibited a nearly 1000-fold improvement in sensitivity for α-thrombin detection compared to our previous optical microscopy method. The superiority of our GPIbα-mediated platforms lies in their stability for α-thrombin detection through protein-protein interactions. By contrast, assays relying on α-thrombin enzymatic activity using substrates face the challenge of a rapid decrease in postsample collection. These results suggested that the MPB of α-thrombin to GPIbα is an ideal mode for clinical α-thrombin detection, particularly in outpatient settings.

Gelatin but not type I collagen promotes bacteria phagocytosis in PMA-treated U937 human lymphoma cells

ABSTRACT Purpose Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. Materials and Methods Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. Results Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. Conclusion This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Treponema pallidum protein Tp47 induced prostaglandin E2 to inhibit the phagocytosis in human macrophages.

During Treponema pallidum (T. pallidum) infection, the host's immune system actively engages in pursuit and elimination of T. pallidum, while T. pallidum skillfully employs various mechanisms to evade immune recognition. Macrophages exhibit incomplete clearance of T. pallidum invitro and the underlying mechanism of how T. pallidum resists the attack of macrophage remains unclear. To investigate the effect of T. pallidum membrane protein Tp47 on the phagocytosis of macrophages. THP-1-derived macrophages were used to investigate the role of Tp47 in the secretion of Prostaglandin E2 (PGE2) in macrophages and the mechanism by which Tp47 induced the production of PGE2, as well as the impact of PGE2 on the macrophage's phagocytosis. Tp47 (1-10 μg/mL) significantly inhibited the phagocytosis of latex beads and T. pallidum in macrophages (p ≤ 0.05). PGE2 production by macrophages could be induced by Tp47, and the phagocytic function of macrophages could be restored using PGE2 antibody. Tp47 produced PGE2 by activating the PERK/NF-κB/COX-2 pathway in macrophages. Inhibitors targeting PERK, NF-κB and COX-2, respectively, reduced the level of PGE2 and restored the phagocytic function of macrophages. Tp47-induced PGE2 production via the PERK/NF-κB/COX-2 pathway contributed to macrophage phagocytosis inhibition, which potentially contributes to immune evasion during the T. pallidum infection.

Clinical assays rapidly predict bacterial susceptibility to monoclonal antibody therapy.

With antimicrobial resistance (AMR) emerging as a major threat to global health, monoclonal antibodies (MAbs) have become a promising means to combat difficult-to-treat AMR infections. Unfortunately, in contrast with standard antimicrobials, for which there are well-validated clinical laboratory methodologies to determine whether an infecting pathogen is susceptible or resistant to a specific antimicrobial drug, no assays have been described that can inform clinical investigators or clinicians regarding the clinical efficacy of a MAb against a specific pathogenic strain. Using Acinetobacter baumannii as a model organism, we established and validated 2 facile clinical susceptibility assays, which used flow cytometry and latex bead agglutination, to determine susceptibility (predicting in vivo efficacy) or resistance (predicting in vivo failure) of 1 newly established and 3 previously described anti-A. baumannii MAbs. These simple assays exhibited impressive sensitivity, specificity, and reproducibility, with clear susceptibility breakpoints that predicted the in vivo outcomes in our preclinical model with excellent fidelity. These MAb susceptibility assays have the potential to enable and facilitate clinical development and deployment of MAbs that generally target the surface of microbes.

Agreement and internal quality assurance of the Neubauer hemocytometer and Makler chamber for human sperm concentration determination.

The Neubauer hemocytometer, as well as the Makler chamber, are devices commonly used in andrology laboratories. The present study aimed to verify if both methods yield comparable results, and whether they can be used interchangeably to determine sperm concentration. Sperm and latex beads concentration measurements were performed with the Neubauer hemocytometer and the Makler chamber. Fixed and proportional biases were estimated, and the method agreement was determined by assessing sperm concentration results with the Bland and Altman plot. The Coefficient of Variation (CV) and relative bias were calculated as an index of precision and accuracy, respectively, by measuring latex beads target concentrations in both chambers. The Makler chamber systematically overestimated the Neubauer hemocytometer concentration measurements by a mean of -7.99%, with limits of agreement (LOA) between -41% to 25.61% (p<0.001). The fixed bias was found for concentration values inferior to 40 x 106/ml range (p<0.001), but not higher concentration results (p>0.05). Measurements with the Neubauer hemocytometer showed the greatest consistency in the study with the CV ranging from 3.01% to 6.67%; while the CV with the Makler chamber ranged from 8.46% to 25.64%. The relative bias for the Neubauer hemocytometer determinations varied from 0.12% to 8.40%, while for the Makler chamber varied from 7.6% to an overestimation of 38.0%. Measurements made with the Makler chamber demonstrated more variability and a higher degree of overestimation. The Makler chamber is a poor substitute to the Neubauer hemocytometer for evaluation of oligozoospermic samples, although both chambers render similar results for highly concentrated samples.

Consequences of mitochondrial DNA depletion in microglial cells

AbstractBackgroundMitochondrial dysfunction is an early and prominent feature of Alzheimer’s disease (AD). We have recently published that lower brain mitochondrial DNA copy number (mtDNAcn) is associated with a greater odds of AD neuropathological change and worse cognitive performance. As it is known that microglial cells play an important role in AD, we want to address how a reduction in mtDNAcn may affect microglial function.Methodsmicroglial BV2 cells were treated with ethidium bromide (etbr) (1ug/ml) for 5 days to achieve a 95% reduction of mitochondrial‐encoded genes expression. Cells were characterized by RNAseq and Seahorse experiments. BV2 cells were challenged with latex beads and 10uM zymosan‐pHrodo for 24 hours and phagocytosis was analyzed by flow cytometry. BV2 cells were stimulated with LPS (100ng/ml) for 3 hours to measure cytokine release by qPCR and ELISA. After BV2 stimulation, fresh media (without LPS or etbr) was conditioned for 6 hours and collected to treat neurons (SH‐SY5Y cells) and human primary astrocytes for 24 hours. RNA and protein samples were collected.ResultsmtDNAcn reduction led to a dramatic reduction in oxidative phosphorylation and a mild reduction in proliferation. RNAseq data showed an increase in the hypoxia signature and cellular senescence, coupled with a reduction in biosynthetic process and response to interferon. No changes were observed in phagocytic capacity. However, after LPS stimulation, etbr‐treated cells showed a reduction in the production of IL1B and IL6 cytokines, suggesting an impairment in the response to challenge. Neurons exposed to media from etbr‐treated BV2 cells showed an increase in MAPT and APOE expression. Samples from astrocytes are currently being analyzed.ConclusionsmtDNAcn reduction impairs mitochondrial respiration, which affects the production of cytokines in response to stimulus and leads to cell senescence in microglial cells. Cytokines released by microglia affected neuronal expression of AD‐related genes.

Performance evaluation of sperm concentration, motility, and morphological analysis for GSA-810 series of sperm quality analysis system.

The performance evaluation of each computer-assisted sperm analysis (CASA) system may provide a basis for the interpretation of clinical results and further improvement of the CASA system. The accuracy of the GSA-810 CASA system was evaluated by detecting latex bead quality control products. The precision of sperm concentration, morphology, and percentages of progressively motile sperm (PR) were evaluated by coefficient of variation (CV). Three samples with sperm concentration of about 100 × 106 /mL were diluted to evaluate the linear range. The detection values of latex beads were within the range of target values. The CVs of sperm concentration and PR were significantly and negatively correlated with sperm concentration (r = -0.561, p = 0.001) and PR value (r = -0.621, p < 0.001), respectively. The R2 values of the linear range of sperm concentration were ≥0.99. There was no significant difference in sperm motility and PR within 1-10 min at 36.5°C ± 0.5°C. The coincidence rates of sperm morphology and sperm head morphology for 36 semen samples analyzed by the GSA-810 system and manual method were 99.40% and 99.67%, respectively. The CVs of the percentage of sperm with abnormal morphology and percentage of sperm with abnormal head morphology were less than 5%. The GSA-810 system can accurately analyze normal semen samples, but the repeatability of the results is poor for oligozoospermia and asthenozoospermia samples. The future CASA system for analyzing sperm morphology should focus on recognizing the middle and tail segments of a spermatozoon.

Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune‑related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Abstract 16891: High Molecular Weight Hyaluronan Inhibits Macrophage Phagocytosis

Background: After a myocardial infarction (MI), macrophages are recruited to the site of injury to begin the process of wound healing. Macrophages are important because they clear cellular debris via phagocytosis. This period of acute inflammation in the heart coincides with extracellular matrix (ECM) remodeling. Hyaluronan (HA), one of the most abundant ECM components, accumulates in a high molecular weight form (HA HMW ). The relationship between HA HMW accumulation and macrophage function in is not fully understood. Goal: To determine the extent to which hyaluronan impacts phagocytosis in polarized macrophages. Methods and Results: Tissue sections were collected from mice one-week post-MI and stained for hyaluronan binding protein (HABP) to assess HA accumulation. A significant increase in HA was seen in both male (n=4, p=0.0031) and female (n=4, p=0.0210) MI hearts compared to sham hearts. Separately, bone marrow was isolated from naïve mice and cells were treated in culture with macrophage colony stimulating factor (M-CSF) for 7-10 days. The resultant macrophages were then polarized into a pro-inflammatory (M1) phenotype using LPS and INF-γ, or pro-reparative phenotype (M2) using IL-4 and IL-13. Macrophage polarization was confirmed via qRT-PCR. In additional experiments, macrophages were polarized for 24 hours and then incubated for 30 min with FITC-labeled, IgG-coated latex beads in the presence or absence of 1 μM HA HMW . Flow cytometry was used to assess phagocytosis of the beads. HA HMW treatment resulted in a significant reduction in the percent of FITC positive cells in M1 female (n=6, p=0.005), M2 female (n=5, p=0.0406), and M2 male (n=5, p=0.0314) macrophage subsets indicating a decrease in phagocytosis. No significant changes were seen in the M0 and M1 male macrophage subsets. Additionally, the mean fluorescence intensity (MFI) measured in the FITC channel was significantly decreased in all the female subsets as well as the M2 male macrophages (n=5-6, p&lt;0.05). No significant changes were seen in the M0 and M1 male subsets. Conclusion: HA HMW impairs opsonin-induced phagocytosis in macrophages. Given that HA HMW accumulates post-MI, these findings may partially explain the incomplete immune response in the failing heart.